Peptide compounds and peptide conjugates for the treatment of cancer through receptor-mediated chemotherapy

ABSTRACT

The present disclosure relates to peptide compounds and conjugate compounds, processes, methods and uses thereof for treating cancer. For example, the compounds can comprise compounds of formulaX1X2X3X4X5GVX6AKAGVX7NX8FKSESY(I)(SEQ ID NO: 1)(X9)nGVX10AKAGVX11NX12FKSESY(II)(SEQ ID NO: 2)YKX13LRRX14APRWDX15PLRDPALRX16X17L(III)(SEQ ID NO: 3)YKX18LRR(X19)nPLRDPALRX20X21L(IV)(SEQ ID NO: 4)IKLSGGVQAKAGVINMDKSESM(V)(SEQ ID NO: 5)IKLSGGVQAKAGVINMFKSESY(VI)(SEQ ID NO: 6)IKLSGGVQAKAGVINMFKSESYK(VII)(SEQ ID NO: 7)GVQAKAGVINMFKSESY(VIII)(SEQ ID NO: 8)GVRAKAGVRNMFKSESY(IX)(SEQ ID NO: 9)GVRAKAGVRN(Nle)FKSESY(X)(SEQ ID NO: 10)YKSLRRKAPRWDAPLRDPALRQLL(XI)(SEQ ID NO: 11)YKSLRRKAPRWDAYLRDPALRQLL(XII)(SEQ ID NO: 12)YKSLRRKAPRWDAYLRDPALRPLL(XIII)(SEQ ID NO: 13)wherein X1 to X21 and n can have various different valuesand wherein at least one protecting group and/or at least one labelling agent is optionally connected to said peptide compound at an N- and/or C-terminal end.

RELATED APPLICATIONS

This present application is a continuation of Ser. No. 15/778,626 filedon May 24, 2018 that is a 35 USC 371 national stage entry ofPCT/CA2016/051379 filed on Nov. 24, 2016 and which claims the benefit ofpriority of U.S. Patent Application Ser. No. 62/259,178 filed on Nov.24, 2015. These documents are hereby incorporated herein by reference intheir entirety.

FIELD OF THE DISCLOSURE

The present disclosure relates to peptide compounds and conjugatecompounds, processes, methods and uses thereof for treating cancer.

BACKGROUND OF THE DISCLOSURE Cancer

According to a recent World Health Organization report (February, 2014),8.2 million patients died from cancer in 2012. Cancer is therefore agrowing health problem in both developing and developed countries. Ithas also been estimated that the number of annual cancer cases willincrease from 14 million in 2012 to 22 million within the next twodecades (WHO, 2014). Currently, the classical treatments for cancer arechemotherapy, radiotherapy and surgery.

Resistance to chemotherapy remains a major cause of failure of cancertreatment. This resistance phenotype results from numerous mechanisms.The “traditional” understanding of multidrug resistance (MDR) and itsdriving mechanisms over-simplifies the complexity of a perturbedcellular cancer network and focuses on several pathways/gene families(Orit, 2013). From that perspective, drug resistance is ratherassociated with the induction of drug efflux, activation of DNA repair,variations in target proteins, decreased drug uptake, alteredmetabolisms, sequestration, and changes in apoptotic pathways (Fodalet,2011; Gillet, 2010). Recently, intratumoural heterogeneity has also beeninferred to be a major facilitator of drug resistance in reference todifferences observed between cancer cells originating within the sametumour. Indeed, many primary human tumours have been found to containgenetically distinct cellular subpopulations reported to be mainly theresult of stochastic processes and microenvironment signals. In additionto the genetic differences or heterogeneity within a tumour, therapeuticresistance can also be caused by several other nongenetic processes,such as epigenetic changes associated with chromatin modification or DNAmethylation (Sanz-Moreno, 2008). One study of these processes wasperformed in a system with a single genetic clone, and concluded thatthere was functional variability among tumour cells (Kreso, 2013;Marusyk, 2013). Clearly, the integration of both genetic and nongeneticassumptions as well as heterogeneity should be included in the design ofnew experimental and computational models to have a better descriptionand ultimately a solution to the problem of MDR.

Multidrug Resistance

Clinical progress in the treatment of primary tumours has been slow. Oneof the problems associated with the treatment of these tumours is theirrelatively weak response to anticancer drugs (Zhou, 2008; Silvia, 2015).The effectiveness of chemotherapy and immunotherapy has been impaired byinherent or acquired MDR phenotype by cancer cells. One of the majormechanisms involved in MDR phenotype involves the expression ofP-glycoprotein (P-gp), a membrane transporter that pumps out variousanticancer drugs from MDR cells. P-gp is also expressed in a largenumber of normal secretory tissues such as kidney, liver and intestine.In humans, it has been reported that P-gp is encoded by two MDR genes(MDR1 and MDR3). Human MDR1 confers the resistance phenotype, whereashuman MDR3 does not. Thus, P-gp may be considered as a “guardian” thatlimits the entry of drugs by expulsing them out of cancer cellspreventing them from reaching cytotoxic concentrations.

Tumour Heterogeneity Intra- and Inter-Tumoural Heterogeneity

Cancer is a devious foe, revealing new complexities just as scientistsfind new ways to tackle them. A recent hope has been put in the newgeneration of “targeted therapeutics” that home in on specific moleculardefects in cancer cells, promising more effective and less toxic therapythan imprecise chemotherapeutic agents (Fisher, 2013). However,researchers are now realizing that they may have previously underestimated one of cancer's oldest and best-known complexity: tumourheterogeneity. This, in part, explains the successes and disappointmentswith targeted therapeutics and should motivate a broader re-examinationof current research strategies.

Tumour heterogeneity refers to the existence of subpopulations of cells,with distinct genotypes and phenotypes that may harbour divergentbiological behaviours, within a primary tumour and its metastases, orbetween tumours of the same histopathological subtype (intra- andinter-tumour, respectively) (Corbin, 2013). With the advent of deepsequencing techniques, the extent and prevalence of intra- andinter-tumour heterogeneity is increasingly acknowledged. There arefeatures of intra-tumour heterogeneity that form part of routinepathologic assessment, but its determination does not yet form part ofthe clinical decision-making process. Nuclear pleomorphism is anotherexample of intra-tumour heterogeneity, which is accounted for in breastcancer grading, for instance. It is also readily apparent to clinicianstreating cancer that there is marked variation in tumour behaviourbetween patients with the same tumour type, and between different tumoursites in the same patient; the latter is usually manifested asdifferential or mixed responses to therapy.

Clonal Evolution as a Model of Tumour Progression and Heterogeneity

A clonal evolutionary model of cancer development was first proposed byNowell (1976) and elaborates upon Darwinian models of naturalselection—that is, genetically unstable cells accumulate geneticalterations, and that selective pressures favour the growth and survivalof variant subpopulations with a biological fitness advantage. Spatialand temporal heterogeneity may permit the tumour as a whole to adapt toa fluctuating tumour microenvironment. In summary, it is argued thatheterogeneous tumours should be viewed as complex ecosystems orsocieties, in which even a minor tumour subpopulation may influencegrowth of the entire tumour and thereby actively maintain tumourheterogeneity (Heppner, 1984; Marusyk, 2010; Bonavia, 2011). In thismodel, subclones occupy various niches within the tumourmicroenvironment and the survival advantage of the tumour ‘society’exceeds those of the individual subpopulation; relationships betweensubclones may be competitive, commensal, or mutualistic for thispurpose.

Clinical Implications of Tumour Heterogeneity

The issue of cancer heterogeneity, including the relationships betweensubpopulations within and between tumour lesions, may have profoundimplications for drug therapy in cancer. Targeted therapy, whichattempts to exploit a tumour's dependence on a critical proliferation orsurvival pathway, has significantly improved patient outcomes in a rangeof solid tumour types, but in the majority of advanced disease cases, itis also apparent that targeted therapeutics do not help all molecularlyselected patients and even when clinical benefit is observed, it isoften of limited duration (Gore, 2011; Diaz, 2012). Tumour heterogeneitymay partly explain these clinical phenomena, and this prompts for thedevelopment of a more efficient platform that circumvents the MDRphenotype.

SUMMARY OF THE DISCLOSURE

Accordingly, a first aspect is a peptide compound having at least 80%sequence identity to a compound chosen from compounds of formula (I),formula (II), formula (III), formula (IV), formula (V), formula (VI),formula (VII), formula (VIII), formula (IX), formula (X), formula (XI)and formula (XII):

X₁X₂X₃X₄X₅GVX₆AKAGVX₇NX₈FKSESY (I) (SEQ ID NO: 1)(X₉)_(n)GVX₁₀AKAGVX₁₁NX₁₂FKSESY (II) (SEQ ID NO: 2)YKX₁₃LRRX₁₄APRWDX₁₅PLRDPALRX₁₆X₁₇L (III) (SEQ ID NO: 3)YKX₁₈LRR(X₁₉)_(n)PLRDPALRX₂₀X₂₁L (IV) (SEQ ID NO: 4)IKLSGGVQAKAGVINMDKSESM (V) (SEQ ID NO: 5) IKLSGGVQAKAGVINMFKSESY (VI)(SEQ ID NO: 6) IKLSGGVQAKAGVINMFKSESYK (VII) (SEQ ID NO: 7)GVQAKAGVINMFKSESY (VIII) (SEQ ID NO: 8) GVRAKAGVRNMFKSESY (IX)(SEQ ID NO: 9) GVRAKAGVRN(Nle)FKSESY (X) (SEQ ID NO: 10)YKSLRRKAPRWDAPLRDPALRQLL (XI) (SEQ ID NO: 11) YKSLRRKAPRWDAYLRDPALRQLL(XII) (SEQ ID NO: 12) YKSLRRKAPRWDAYLRDPALRPLL (XIII) (SEQ ID NO: 13)

wherein

-   -   X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄,        X₁₅, X₁₈ and X₁₉ are independently chosen from any amino acid;    -   X₁₆, X₁₇, X₂₀ and X₂₁ are independently chosen from Q, P, Y, I        and L;    -   n is 0, 1, 2, 3, 4 or 5,    -   when X₉ is present more than once, each of said X₉ is        independently chosen from any amino acid;    -   when X₁₉ is present more than once, each of said X₉ is        independently chosen from any amino acid;

and wherein at least one protecting group and/or at least one labellingagent is optionally connected to said peptide at an N- and/or C-terminalend.

In a further aspect, there is provided peptide compounds that are infact any peptide compounds described in the present disclosure, to whichabout 5 or 6 amino acids have been omitted or removed.

In a further aspect, there is provided peptide compounds that are infact compounds comprising at least 5 or at least 6 consecutive aminoacids as defined in the previously presented peptide compounds.

In a further aspect, there is provided a peptide compound comprising acompound chosen from compounds of formula (I), formula (II), formula(III), formula (IV), formula (V), formula (VI), formula (VII), formula(VIII), formula (IX), formula (X), formula (XI) and formula (XII):

(SEQ ID NO: 1) X₁X₂X₃X₄X₅GVX₆AKAGVX₇NX₈FKSESY (I) (SEQ ID NO: 2)(X₉)_(n)GVX₁₀AKAGVX₁₁NX₁₂FKSESY (II) (SEQ ID NO: 3)YKX₁₃LRRX₁₄APRWDX₁₅PLRDPALRX₁₆X₁₇L (III) (SEQ ID NO: 4)YKX₁₈LRR(X₁₉)_(n)PLRDPALRX₂₀X₂₁L (IV) (SEQ ID NO: 5)IKLSGGVQAKAGVINMDKSESM (V) (SEQ ID NO: 6) IKLSGGVQAKAGVINMFKSESY (VI)(SEQ ID NO: 7) IKLSGGVQAKAGVINMFKSESYK (VII) (SEQ ID NO: 8)GVQAKAGVINMFKSESY (VIII) (SEQ ID NO: 9) GVRAKAGVRNMFKSESY (IX)(SEQ ID NO: 10) GVRAKAGVRN(Nle)FKSESY (X) (SEQ ID NO: 11)YKSLRRKAPRWDAPLRDPALRQLL (XI) (SEQ ID NO: 12) YKSLRRKAPRWDAYLRDPALRQLL(XII) (SEQ ID NO: 13) YKSLRRKAPRWDAYLRDPALRPLL (XIII)

wherein

-   -   X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄,        X₁₅, X₁₈ and X₁₉ are independently chosen from any amino acid;    -   X₁₆, X₁₇, X₂₀ and X₂₁ are independently chosen from Q, P, Y, I        and L;    -   n is 0, 1, 2, 3, 4 or 5,    -   when X₉ is present more than once, each of said X₉ is        independently chosen from any amino acid;    -   when X₁₉ is present more than once, each of said X₉ is        independently chosen from any amino acid;

and wherein at least one protecting group and/or at least one labellingagent is optionally connected to said peptide at an N- and/or C-terminalend.

In a further aspect disclosed herein is a conjugate compound having theformula of A-(B)_(n),

wherein

-   -   n is 1, 2, 3 or 4;    -   A is a peptide compound as defined in the present disclosure,        wherein said peptide is optionally protected by a protecting        group; and    -   B is at least one therapeutic agent, wherein B is connected to        A.

In a further aspect disclosed herein is a conjugate compound having theformula of A-(B)_(n),

wherein

-   -   n is 1, 2, 3 or 4;    -   A is a peptide compound as defined in the present disclosure,        wherein said peptide is optionally protected by a protecting        group; and    -   B is at least one therapeutic agent, wherein B is connected to A        at a free amine of said peptide compound, at an N-terminal        position of said peptide compound, at a free —SH of said peptide        compound, or at a free carboxyl of said peptide compound.

A further aspect disclosed herein is a conjugate compound having theformula of A-(B)_(n),

wherein

-   -   n is 1, 2, 3 or 4;    -   A is a peptide compound as defined in the present disclosure,        wherein said peptide is optionally protected by a protecting        group; and    -   B is at least one therapeutic agent, wherein B is connected to A        at a free amine of a lysine residue of said peptide compound,        optionally via a linker, or at an N-terminal position of said        peptide compound, optionally via a linker.

In a further aspect, there is provided a process for preparing theconjugate compound herein disclosed, the process comprising:

-   -   reacting a linker together with said therapeutic agent so as to        obtain an intermediate;    -   optionally purifying said intermediate;    -   reacting said intermediate together with said peptide compound        so as to obtain said conjugate compound in which said        therapeutic agent is connected to said peptide compound via said        linker; and    -   optionally purifying said conjugate compound;        wherein the therapeutic agent is connected to the peptide        compound at a free amine of a lysine residue or at an        N-terminal; and wherein the peptide compound comprises 1, 2, 3        or 4 therapeutic agent molecules connected thereto.

In another aspect, there is provided a method of treating a cancercomprising administrating a therapeutically effective amount of at leastone compound herein disclosed to a subject in need thereof.

In another aspect, there is provided a method of treating a cancerinvolving sortilin expression comprising contacting at least one cancercell expressing sortilin with at least one compound as defined herein.

In another aspect, there is provided a method of treating a diseaseinvolving sortilin expression comprising administering to a subject inneed thereof a therapeutically effective amount of at least one compoundas defined herein.

In another aspect, there is provided a use of a compound disclosedherein for treating a cancer.

Another aspect is a library comprising at least two of compounds hereindisclosed.

Another aspect is a liposome, graphene or nanoparticle comprising atleast one compound as defined in the present disclosure.

Another aspect is a liposome, graphene or nanoparticle coated with atleast one compound as defined in the present disclosure.

Another aspect is a liposome, graphene or nanoparticle that is loadedwith at least one of therapeutic agent or siRNA and the liposome iscoated with at least one compound as defined in the present disclosure.

Another aspect relates to a drug delivery system comprising such aliposome, graphene or nanoparticle as defined in the present disclosure.

Another aspect is the use of such liposome, graphene or nanoparticle asdefined in the present disclosure, in a drug delivery system.

A further aspect relates to a multimer comprising two or more compoundsherein disclosed.

BRIEF DESCRIPTION OF THE FIGURES

Further features and advantages of the disclosure will become morereadily apparent from the following description of specific embodimentsas illustrated by way of examples in the appended figures wherein:

FIG. 1 shows the interaction between Katana peptide and sortilin usingsurface plasmon resonance. Biotinylated Katana peptide was immobilizedon a streptavidin sensor chip. A. Soluble sortilin was then injectedover the immobilized peptide. The surface plasmon resonance signal wasthen monitored over time.

FIG. 2 is a prior art representation of the efflux pump, P-glycoprotein(P-gp or MDR1) at the cell surface. The efflux pump, P-gp or MDR1,associated with multidrug resistance is highly expressed at the cellsurface of many cancer cells and various tissues.

FIG. 3 shows the immunodetection of sortilin in human cancer cell lines.Equal amounts of protein from human cancer cell lysates were separatedby gel electrophoresis. After electrophoresis, the proteins weretransferred to PVDF membrane and sortilin was immunodetected using amonoclonal antibody directed against this sortilin receptor. Sortilincould be visualized by a secondary antibody directed against mouse IgGlinked to horseradish peroxidase and chemiluminescent reagents.

FIG. 4 is a schematic representation of (A) an anticancer drugconjugated to the Katana peptide and (B) a phytochemical conjugated tothe Katana peptide.

FIG. 5 shows the structures of drug-Katana peptide conjugates. Examplesof Katana peptide conjugates with docetaxel (A) and doxorubicin (B) andof a phytochemical-Katana peptide conjugate with curcumin (C). Differentnumbers (n=1 to 4) of anticancer agent or phytochemical molecules can beincorporated on the N-terminal and lysines of the Katana peptide.

FIG. 6 shows the uptake of unconjugated drugs and of Katana-peptide drugconjugates in MDCK cells transfected with MDR1. 6A. MDCK-MDR1 cells wereincubated with 50 nM of either [³H]-Docetaxel or [¹²⁵I]-Katanapeptide-Docetaxel conjugate (KBA105) for 1 hr at 37° C. in the presenceor absence of the P-gp (MDR1) inhibitor Cyclosporin A (CsA) (10 μM). 6B.MDCK-MDR1 cells were incubated with 50 nM of either [¹⁴C]-Doxorubicin or[¹²⁵I]-Katana peptide-Doxorubicin conjugate (KBB106) for 1 hr at 37° C.in the presence or absence of CsA (10 μM). After the incubation, cellswere washed and radioactivity accumulated in cells was quantified. Theresults were expressed in terms of uptake fmol/mg of protein.

FIG. 7 shows the uptake of radiolabeled Docetaxel and Katana conjugate(KBA105) in SKOV3 (FIG. 7A) and SK-MEL28 (FIG. 7B) cancer cells. Cellswere incubated for up to 2 hrs with radiolabeled compounds. Afterincubation, cells were washed 3-times with PBS and accumulatedradioactivity was then quantified. Results were expressed in terms ofuptake (fmol/mg of protein) as function of time.

FIG. 8 shows the uptake of Katana Doxorubicin conjugate (KBB106) isreduced in cells where sortilin expression is reduced or by sortilinligands. A. Ovarian cancer cells were transfected with sortilin siRNA(siSortilin) for 24 hrs and then incubated with KBB106. Accumulation ofKBB106 was monitored by the released of fluorescent Doxorubicin as afunction of time. B. Ovarian cancer cells were incubated with KBB106 inthe presence or absence of sortilin ligand (Katana peptide,progranulin). KBB106 accumulation was estimated by the released offluorescent Doxorubicin.

FIG. 9 shows that the conjugated-Docetaxel induces a better andsustained cell death of ovarian cancer cells. 9A. Effect of KBA105 onovarian (SKOV3) cancer cells apoptosis was compared to that ofDocetaxel. After incubation, cells were washed and stained for AnnexinV. 9B. Conjugated Docetaxel induces a stronger apoptosis thanunconjugated Docetaxel. This level of apoptosis is similar to thatmeasure for Docetaxel in the presence of the P-gp (MDR1) inhibitorCyclosporine A (CsA). 9C. Dose-dependent increased apoptotic potentialby KBA015 compared to Docetaxel. Cells were incubated for 5 hrs withincreasing concentration of KBA105 or Docetaxel and levels of apoptosiswas then determined. Results are expressed in terms of apoptosispercentage as a function of drug concentration.

FIG. 10 shows the effect of Katana-drug conjugates on ovarian, skin andbreast cancer cells apoptosis. Cancer cells were incubated for 5 hrswith 2 μM of Katana-drug conjugates or unconjugated Docetaxel orCabazitaxel. After incubation, cells were washed and stained for AnnexinV. Results are expressed in terms of apoptosis percentage for thedifferent drugs.

FIG. 11 shows the reversal of Katana-drug conjugate effect on cancercell apoptosis by unconjugated peptide and sortilin ligands. Ovarian(SKOV3) cancer cells were incubated for 5 hrs with 2 μM of DocetaxeKatana-drug conjugates KBA-105 (11A) and KBA-201 (11B) in the absence orpresence of free peptide, neurotensin (NT) and progranulin. Afterincubation, cells were washed and stained for Annexin V. Results areexpressed in terms of apoptosis percentage of SKOV3 cancer cells.

FIG. 12 shows increased anti-migratory effects of Docetaxel conjugatedto the Katana-peptide on ovarian cancer cells.

FIG. 13 is a series of graphs providing validation of the Katanaplatform delivery and evidence for receptor-mediated internalization ofconjugated-Docetaxel in ovarian cancer cells. Co-incubation ofneurotensin (13A) or the Katana-peptide (free peptide) (13B) withdocetaxel does not alter the effect of docetaxel on cell migration. Incontrast, the addition of the free peptide or neurotensin toKatana-docetaxel conjugate reverses its effects on SKOV3 migration. 13C.Results show that sortilin gene silencing with specific sortilin siRNAreverses the effect of the Docetaxel conjugate (KBA105) on ovariancancer cells migration.

FIG. 14 shows the effect of Katana-drug conjugates on ovarian, skin andbreast cancer cell migration. The cells were incubated for 2 hrs withdifferent Katana Taxane conjugates and cell migration was thenperformed. All conjugates strongly affected the migration of the variouscancer cells.

FIG. 15 shows of the effect of Katana-drug conjugates on ovarian cancercell migration. 15A. Cells were incubated for 2 hrs with increasingconcentration of Doxorubicin or conjugated Doxorubicin (KBB106) and cellmigration was performed. 15B. Results show that Neurotensin (NT) and theKatana peptide (KBP106) reversed the effect of KBB106 on ovarian cancercell (ES-2) migration. 15C. Progranulin (PGR), a sortilin ligand, alsoreversed the effect of the Doxorubicin conjugate (KBB106) on ovariancancer cell (ES-2) migration.

FIG. 16 is a representation of relative sortilin expression in ovariancancers and brain tumors. 16A. Sortilin was detected by Western blot inhuman normal tissues and ovarian cancer biopsies (from Ghaemimanesh etal. 2014). Results show sortilin is overexpressed in ovarian cancerbiopsies compared to normal tissues. Sortilin gene levels were estimatedin cDNA samples (Origene; Rockville, Md., USA) from patients withdifferent brain (16B) and ovarian (16C) tumor grades.

FIG. 17 is a prior art representation of sortilin (SORT1) expression inhuman cancers from the Human Protein Atlas website(http://www.proteinatlast.org).

FIG. 18 shows the expression of sortilin in ovarian tissues byimmunohistochemistry (IHC). 18A. Sortilin is overexpressed in primaryovarian tumor and in ovarian metastasis and almost undetectable innon-malignant healthy ovarian tissue. 18B. Sortilin was also detected byIHC in a series of normal ovarian tissue, benign tumors, borderlinetumors, malignant tumors as well as in metastases from ovarian cancers.Results indicate that sortilin expression increased as a function oftheir malignant phenotypes and is higher in ovarian metastases.

FIG. 19 is a representation of pharmacokinetics of the Docetaxel-Katanapeptide conjugate (KBA-105). Mice were injected with radiolabeledconjugate at 5 mg/kg. Plasma was collected at different time points,radioactivity was counted and plasma levels were plotted as a functionof time. Pharmacokinetic parameters were then extracted from the curveusing the «PK solutions» software.

FIG. 20 is a graph showing concentration of unconjugated and conjugatedDocetaxel as a function of time. Mice were injected iv with radiolabeled[³H]-Docetaxel or radiolabeled [¹²⁵I]-KBA105 at an equivalent dose ofDocetaxel (Docetaxel=2.2 mg/kg; KBA105=5 mg/kg). Plasma samples werecollected at different time points and radioactivity quantified. Resultsare expressed in terms of KBA105 or Docetaxel concentration in plasma asa function of time. Area under the curve for the time period (AUC1-24)was estimated for KBA105 and Docetaxel using GraphPad Prism software.Ratio between AUC1-24 for KBA105 and Docetaxel was calculated to bearound 35.

FIG. 21 shows the tissue distribution of [¹²⁵I]-Katanapeptide-Docetaxel. CD-1 mice were injected with the radiolabeledconjugate at 5 mg/kg via iv bolus injections (21A) or 2.2 mg ofradiolabeled Docetaxel (21B). At the indicated times (1 h, 6 h, 24 h)mice were sacrificed and perfused with saline for 8 min. Tissues werethen collected and radioactivity was measured. Results are expressed interms of ng/g of tissue.

FIG. 22 shows the effect of KBA105 and Docetaxel on ovarian subcutaneoustumors. Mice were implanted in the flank with SKOV3 cancer cells. Tumorgrowth was monitored by luminescence using the Near Infrared imagingsystem from Carestream. When tumors reached similar luminescence, micewere treated with Docetaxel (22A) or KBA105 (22B) at an equivalent doseof Docetaxel (10 mg/kg/week).

FIG. 23 shows the luminescence quantitation of FIG. 22. Results areexpressed in terms of luminescence intensity as a function ofpost-treatment days.

FIG. 24 is a graph showing the body weight of mice treated withDocetaxel and KBA105 monitored over treatment days. Results indicatethat at the dose administered Docetaxel has a strong effect on the bodyweight. In contrast, at the equivalent dose of Docetaxel, KBA105 has noeffect on the body weight of mice. These results indicate that at anequivalent dose of Docetaxel, KBA105 is better tolerated.

FIG. 25 shows the effect of the Doxorubicin conjugate (KBB106) andunconjugated Doxorubicin on ovarian subcutaneous tumors. 25A. Mice wereimplanted in the flank with ES-2 ovarian cancer cells. Tumor growth wasmeasured using a caliper. When tumors reached a tumor volume of about150 mm³, mice were treated with Doxorubicin or KBB106 at an equivalentdose of Doxorubicin (6 mg/kg/week). 25B. Results show tumor volumeincrease at Day 14 post-treatment in mice treated with Doxorubicin orKBB106 compared to the vehicle group. 25C. Because of KBB106 efficacyand tolerability, treatments of mice with the conjugate were continuedup to Day 66 post-treatment.

FIG. 26 shows the effect of the Doxorubicin conjugate (KBB106) onovarian subcutaneous tumors. 26A. Mice were implanted in the flank withSKOV3 ovarian cancer cells. When tumors reached a volume of about 150mm³, mice were treated with KBB106 (18 mg/kg/week) as indicated by thearrows. Results show tumor volume progression (initial tumor volume atD0 was subtracted) in mice treated with KBB106 compared to the vehiclegroup. 26B. Body weight of mice was monitored during the treatments.Absence of body weight loss in mice treated with KBB106 suggests thetreatments were well tolerated.

FIG. 27 shows the effect of the Docetaxel conjugate (KBA106) andunconjugated Docetaxel on breast subcutaneous tumors. 27A. Mice wereimplanted in the flank with MDA-MB231 breast cancer cells expressingluciferase. Tumor growth by luminescence was visualized using an in vivoimaging system from Carestream. Mice were treated with vehicle,Docetaxel (15 mg/kg/week) or KBA106 (50 mg/kg/week). Initialluminescence intensity was subtracted. 27B. Results show the residualtumor burden evaluated by the remaining luminescence associated withcancer cells at Day 74. No value was available (N/A) for the micetreated with the vehicle since all mice were then sacrificed because thesize of their tumors reached the endpoint limit. 27C. Tumor luminescenceresults were expressed in terms of percentage of progression on D74post-treatment compared to initial tumor luminescence. In the Docetaxelgroup, the tumor luminescence increased by 100% whereas in the KBA106treated group the luminescence decreased by 100%. In the KBA106 group,no luminescence was detectable on Day 74 post-treatment suggesting noresidual tumors in these mice.

FIG. 28 shows the effect of the Curcumin conjugate (KBC106) andunconjugated Curcumin on cancer cell proliferation. Breast cancer cells(MDA-MB231) were incubated with increasing concentrations of KBC106 orcurcumin. After 72 hrs, thymidine incorporation assay was performed toassess the anti-proliferative properties of both molecules. IC50 valueswere extracted from the anti-proliferative curves. Results show thatKBC106 has a stronger anti-proliferation activity (about 100-fold)against these breast cancer cells compared to unconjugated curcumin.

FIG. 29 shows the preferential sortilin dependent uptake of Curcuminconjugate (KBC106) in ES-2 ovarian cancer cells. A. Cancer cells wereincubated with KBC106 or curcumin. Uptake for both molecules wasevaluated by fluorescent live imaging. Higher accumulation offluorescence was detected for KBC1006 indicating a better cellinternalization for the conjugate. B. Uptake of KBC106 was monitored byin vitro fluorescence imaging in the presence of sortilin ligandsNeurotensin (NT) and progranulin as well as free Katana peptide.Accumulation of KBC106 in ovarian cancer cells was inhibited by sortilinligands. C. Results of KBC106 accumulation were expressed in terms ofKBC106 fluorescence intensity in the presence or absence of sortilinligands. Data demonstrate pharmacological competition of KBC106 uptakeby sortilin ligands.

FIG. 30 is a demonstration that the Curcumin conjugate (KBC106) inducescancer cell apoptosis.

FIG. 31 is a demonstration that the Curcumin conjugate (KBC106) inhibitstumor growth of endometrial (MES) subcutaneous tumors. Mice wereimplanted in the flank with endometrial (MES) cancer cells. Tumor growthwas measured using a caliper. When tumors reached a volume of around 150mm³, mice were treated with KBC106 at 60 mg/kg/twice a week.

DETAILED DESCRIPTION OF THE DISCLOSURE

The term “peptide compounds” or “Katana peptides”, “Katana BiopharmaPeptide” or “KBP” as used herein refers, for example, to peptidesderived from bacterial proteins or from ligands of receptors that targetreceptors expressed on cancer cells including multidrug resistant cancercells. For example, the peptide compounds can be derived from bacterialproteins involved in cell penetration or from sortilin ligands, forexample progranulin and neurotensin. In certain embodiments, peptidecompounds are connected (for example via a covalent bond, an atom or alinker) to at least one therapeutic agent (such as an anticancer agentor a phytochemical), thereby forming a conjugate compound that can beused, for example, for treating a cancer. In certain other embodiments,peptide compounds can be used at the surface of liposomes. For example,the peptide compounds can be used for coating liposomes or nanoparticlesthat can be loaded with at least one therapeutic agent (such as ananticancer agent or phytochemical, or genes or siRNA).

The term “Katana Biopharma Peptide Family 1 peptide compounds” or “KBPFamily 1 peptide compounds” refers to peptide compounds derived frombacterial cell penetrant proteins. For example, KBP Family 1 peptidecompounds can be derived from a protein having an amino acid sequence ofIKLSGGVQAKAGVINMDKSESM (SEQ ID NO: 5). Non limiting examples of KBPFamily 1 peptide compounds are shown below:

Amino acid sequences IKLSGGVQAKAGVINMDKSESM - KBP-101Formula (V) (represented by SEQ ID NO: 5)Succinyl-IKLSGGVQAKAGVINMFKSESY - KBP-102Formula (XXXVI) (comprises SEQ ID NO: 6 wherein asuccinyl group is attached at the N-terminal end)IKLSGGVQAKAGVINMFKSESYK(Biotin) - KBP-103Formula (XXXVII) (comprises SEQ ID NO: 7 wherein abiotin molecule is connected thereto at the C-terminal end)GVQAKAGVINMFKSESY - KBP-104 Formula (VIII) (represented by SEQ ID NO: 8)Acetyl-GVRAKAGVRNMFKSESY - KBP-105Formula (XXXVIII) (represented by SEQ ID NO: 14)Acetyl-GVRAKAGVRN(Nle)FKSESY - KBP-106Formula (XXXIX) (represented by SEQ ID NO: 15)

As used herein, the peptide compound KBP-101 is represented by the aminoacid sequence of IKLSGGVQAKAGVINMDKSESM (SEQ ID NO: 5).

As used herein, the peptide compound KBP-102 is represented by the aminoacid sequence of Succinyl-IKLSGGVQAKAGVINMFKSESY that comprises thepeptide sequence of SEQ ID NO: 6 wherein a succinyl group is attachedthereto at the N-terminal end.

As used herein, the peptide compound KBP-103 is represented by the aminoacid sequence of IKLSGGVQAKAGVINMFKSESYK(Biotin) that comprises thepeptide sequence of SEQ ID NO: 7 wherein a biotin molecule is connectedthereto at the C-terminal end.

As used herein, the peptide compound KBP-104 is represented by the aminoacid sequence of GVQAKAGVINMFKSESY (SEQ ID NO: 8).

As used herein, the peptide compound KBP-105 is represented by the aminoacid sequence of Acetyl-GVRAKAGVRNMFKSESY (SEQ ID NO: 14).

As used herein, the peptide compound KBP-106 is represented by the aminoacid sequence of Acetyl-GVRAKAGVRN(Nle)FKSESY (SEQ ID NO: 15).

The term “Katana Biopharma Peptide Family 2 peptide compounds” or “KBPFamily 2 peptide compounds” refers to peptides derived from sortilinligands, progranulin and neurotensin. For example, peptides can bederived from human, rat or mouse progranulin. For example, KBP Family 2peptide compounds can be derived from human progranulin, for examplehaving the amino acid sequence KCLRREAPRWDAPLRDPALRQLL (SEQ ID NO: 19),from rat progranulin, for example having the amino acid sequenceKCLRKKTPRWDILLRDPAPRPLL (SEQ ID NO: 20), from mouse progranulin, forexample having the amino acid sequence KCLRKKIPRWDMFLRDPVPRPLL (SEQ IDNO: 21), or from neurotensin, for example having an amino acid sequenceXLYENKPRRPYIL (SEQ ID NO: 22). Non limiting examples of KBP Family 2peptide compounds are shown below:

Amino acid sequences Acetyl-YKSLRRKAPRVVDAPLRDPALRQLL - KBP-201Formula (XXXX) (represented by SEQ ID NO: 16)Acetyl-YKSLRRKAPRVVDAYLRDPALRQLL - KBP-202Formula (XXXXI) (represented by SEQ ID NO: 17)Acetyl-YKSLRRKAPRVVDAYLRDPALRPLL - KBP-203Formula (XXXII) (represented by SEQ ID NO: 18)

As used herein, the peptide compound KBP-201 is represented by the aminoacid sequence of Acetyl-YKSLRRKAPRWDAPLRDPALRQLL (SEQ ID NO: 16).

As used herein, the peptide compound KBP-202 is represented by the aminoacid sequence of Acetyl-YKSLRRKAPRWDAYLRDPALRQLL (SEQ ID NO: 17).

As used herein, the peptide compound KBP-203 is represented by the aminoacid sequence of Acetyl-YKSLRRKAPRWDAYLRDPALRPLL (SEQ ID NO: 18).

The term “sortilin” as used herein refers to a neuronal type-1 membraneglycoprotein, encoded by the SORT1 gene, belonging to the VacuolarProtein Sorting 10 protein (Vps10) family of receptors. Sortilin (alsoknown as the neurotensin receptor 3) is expressed abundantly in thecentral and peripheral nervous systems and is also expressed in othertypes of tissues. For example, the expression of sortilin is upregulatedin a number of cancers including for example ovarian, breast, colon andprostate cancer. Sortilin can exist in two forms, a full-length form(110 kDa) and a truncated form (95 kDa), corresponding to its largeluminal domain (or ectodomain), which has been previously detected inthe supernatant medium from sortilin-overexpressing cells (Navarro etal., 2002) The peptide compounds and conjugate compounds hereindescribed can have a high binding affinity to sortilin and thus canspecifically target cancer cells expressing or overexpressing sortilin.

The term “compound” as used in the present document refers to compoundsof formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X),(XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX),(XXI), (XXII), (XXIII), (XXIV) (XXV), (XXVI), (XXVII), (XXVIII), (XXIX),(XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII),(XXXVIII), (XXXIX), (XXXX), (XXXXI) or (XXXXII) or to pharmaceuticallyacceptable salts, solvates, hydrates and/or prodrugs of these compounds,isomers of these latter compounds, or racemic mixtures of these lattercompounds, and/or to composition(s) made with such compound(s) aspreviously indicated in the present disclosure. The expression“compound” also refers to mixtures of the various compounds hereindisclosed.

Compounds of the present disclosure include prodrugs. In general, suchprodrugs will be functional derivatives of these compounds which arereadily convertible in vivo into the compound from which it isnotionally derived. Prodrugs of the compounds of the present disclosuremay be conventional esters formed with available hydroxy, or aminogroup. For example, an available OH or nitrogen in a compound of thepresent disclosure may be acylated using an activated acid in thepresence of a base, and optionally, in inert solvent (e.g. an acidchloride in pyridine). Some common esters which have been utilized asprodrugs are phenyl esters, aliphatic (C₈-C₂₄) esters, acyloxymethylesters, carbamates and amino acid esters. In certain instances, theprodrugs of the compounds of the present disclosure are those in whichone or more of the hydroxy groups in the compounds is masked as groupswhich can be converted to hydroxy groups in vivo. Conventionalprocedures for the selection and preparation of suitable prodrugs aredescribed, for example, in “Design of Prodrugs” ed. H. Bundgaard,Elsevier, 1985.

Compounds of the present disclosure include radiolabeled forms, forexample, compounds labeled by incorporation within the structure ²H, ₃H,¹⁴C, ¹⁵N, or a radioactive halogen such as ¹²⁵I. A radiolabeled compoundof the compounds of the present disclosure may be prepared usingstandard methods known in the art.

The expression “derivative thereof” as used herein when referring to acompound means a derivative of the compound that has a similarreactivity and that could be used as an alternative to the compound inorder to obtain the same desired result.

The term “cancer” as used herein means a primary or a secondary cancerand includes a non-metastatic cancer and/or a metastatic cancer.Reference to cancer includes reference to cancer cells. For example, thecancer is ovarian cancer, brain cancer, breast cancer, melanoma,colorectal cancer, glioblastoma, liver cancer, lung cancer, prostatecancer, cervical cancer, head cancer, gastric cancer, kidney cancer,endometrial cancer, testis cancer, urothelial cancer, acutelymphoblastic leukemia, acute myeloid leukemia, Hodgkin lymphoma,neuroblastoma, non-Hodgkin lymphoma, soft tissue cancer, bone sarcoma,thyroid cancer, transitional cell bladder cancer, Wilm's tumour, glioma,pancreatic cancer or spleen cancer. The term “cancer” as used hereinalso comprises any cancer involving expression of sortilin.

The term “therapeutic agent” as used herein means an agent capable ofproducing a therapeutic effect by inhibiting, suppressing or reducing acancer (e.g., as determined by clinical symptoms or the amount ofcancerous cells) in a subject as compared to a control. Examples oftherapeutic agents include for example anticancer agents andphytochemicals.

The term “anticancer agent” as used herein means an agent capable ofcausing toxicity in cancer cells. For example, taxanes, which arederived from the bark of the Pacific yew tree Taxus brevifolia, can beused as anticancer agents. Taxanes include for example paclitaxel,docetaxel and cabazitaxel. Other anticancer agents include for exampleanthracycline compounds which work by intercalating DNA. For example,anthracyclines include doxorubicin and daunorubicin.

The term “docetaxel” or “doce” as used herein means an anticancer agenthaving the structure:

or pharmaceutically acceptable salts, solvates or prodrugs thereof aswell as mixtures thereof. For example, docetaxel can be conjugated to apeptide compound of the present disclosure via the oxygen atom attachedto the carbon atom at position 2 of its side chain. Docetaxel can beconnected to the peptide compound directly or via a linker.

The term “doxorubicin” or “doxo” as used herein means an anticanceragent having the structure:

or pharmaceutically acceptable salts, solvates or prodrugs thereof aswell as mixtures thereof. For example, doxorubicin can be conjugated toa peptide compound of the present disclosure via the oxygen atomattached to the carbon atom at position 14. Doxorubicin can be connectedto the peptide compound directly or via a linker.

The term “cabazitaxel” or “cab” as used herein means an anticancer agenthaving the structure:

or pharmaceutically acceptable salts, solvates or prodrugs thereof aswell as mixtures thereof. For example, cabazitaxel can be conjugated toa peptide compound of the present disclosure via the oxygen atomattached to the carbon atom at position 2 of its side chain. Cabazitaxelcan be connected to the peptide compound directly or via a linker.

The term “phytochemical” as used herein means chemical compounds thatoccur naturally in plants and that can be used for treating a cancer.Examples of phytochemicals include for example Curcumin, Genistein,Resveratrol, Epigallocatechin-(3)-gallate (EGCG), Piperine,Sulforaphane, Quercetin, lupeol and ß-Carotene. Curcumin(diferuloylmethane) is a yellow pigment present in the spice turmeric(Curcuma longa) that has been associated with antioxidant,anti-inflammatory, anticancer, antiviral, and antibacterial activitiesas indicated by over 6,000 citations (Hosseini, 2015). Otherphytochemicals that can be used include, without limitation, those shownbelow:

Alkaloids Monoterpenes Chlorogenic acid Geraniol Theobromine LimoneneTheophylline Organosulfides Anthocyanins Allicin Cyanidin GlutathioneMalvidin Indole-3-Carbinol Carotenoids Isothiocyanates Beta-CaroteneSulforaphane Lutein Other Lycopene Phytochemicals CoumestansDamnacanthal Flavan-3-Ols Digoxin Flavonoids Phytic acid EpicatechinPhenolic Acids Catechins Capsaicin Hesperidin Ellagic Acid IsorhamnetinGallic acid Kaempferol Rosmarinic acid Myricetin Tannic Acid NaringinPhytosterols Nobiletin Beta-Sitosterol Proanthocyanidins SaponinsQuercetin Stylbenes Rutin Pterostilbene Tangeretin ResveratrolHydroxycinnamic Triterpenoids Acids Ursolic acid Chicoric acidXanthophylls Coumarin Astaxanthin Ferulic acid Beta-CryptoxanthinScopoletin Isoflavones Daidzein Genistein Lignans Silymarin MonophenolsHydroxytyrosol

The term “curcumin” or “cur” as used herein means a phytochemical havingthe structure:

or pharmaceutically acceptable salts, solvates or prodrugs thereof aswell as mixtures thereof. For example, curcumin can be conjugated to apeptide compound of the present disclosure via an oxygen atom of itsphenol groups. Curcumin can be connected to the peptide compounddirectly or via a linker.

The term “conjugate compounds” or “peptide-drug conjugates” as usedherein refers to compounds comprising a peptide compound hereindisclosed connected to at least one therapeutic agent, optionally via alinker. Conjugate compounds can comprise, for example, 1, 2, 3 or 4molecules of a therapeutic agent connected thereto. These 1-4 moleculesof therapeutic agent can be the same or different i.e. up to fourdifferent therapeutic agents could be connected to the peptides. Thetherapeutic agent(s) are connected to the peptide via at least onecovalent bond, at least one atom or at least one linker. Conjugatecompounds can be used in the treatment of a cancer. Examples ofconjugate compounds include, without limitation, the conjugate compoundsshown below:

Products Amino acid sequences Docetaxel-conjugates KBA-102 (3:1)Succinyl-IK(docetaxel)LSGGVQAK(docetaxel)AGVINMFK(docetaxel)SESY -Formula (XIX) that comprises the peptide compound having SEQ ID NO: 6wherein each lysine residue has a docetaxel molecule connected thereto;and wherein a succinyl group is attached thereto at the N-terminal endKBA-104 (2:1) GVQAK(docetaxel)AGVINMFK(docetaxel)SESY - Formula (XV)that comprises the peptide compound having SEQ ID NO: 8 wherein eachlysine residue has a docetaxel molecule connected thereto; KBA-105 (2:1)Acetyl-GVRAK(docetaxel)AGVRNMFK(docetaxel)SESY - Formula (XX) thatcomprises the peptide compound having SEQ ID NO: 14 wherein each lysineresidue has a docetaxel molecule connected thereto KBA-106 (2:1)Acetyl-GVRAK(docetaxel)AGVRN(Nle)FK(docetaxel)SESY - Formula (XXI) thatcomprises the peptide compound having SEQ ID NO: 15 wherein each lysineresidue has a docetaxel molecule connected thereto KBA-201 (2:1)Acetyl-YK(docetaxel)SLRRK(docetaxel)APRWDAPLRDPALRQLL - Formula (XXII)that comprises that comprises the peptide compound having SEQ ID NO: 16wherein each lysine residue has a docetaxel molecule connected theretoDoxorubicin-conjugates KBB-104 (2:1)GVQAK(doxorubicin)AGVINMFK(doxorubicin)SESY - Formula (XXIII) thatcomprises the peptide compound having SEQ ID NO: 8 wherein each lysineresidue has a doxorubicin molecule connected thereto KBB-106 (2:1)Acetyl-GVRAK(doxorubicin)AGVRN(Nle)FK(doxorubicin)SESY - Formula (XXVI)that comprises the peptide compound having SEQ ID NO: 15 wherein eachlysine residue has a doxorubicin molecule connected thereto KBB-201(2:1) Acetyl-YK(doxorubicin)SLRRK(doxorubicin)APRWDAPLRDPALRQLL -Formula (XXVII) that comprises the peptide compound having SEQ ID NO: 16wherein each lysine residue has a doxorubicin molecule connected theretoCurcumin-conjugates KBC-106 (2:1)Acetyl-GVRAK(curcumin)AGVRN(Nle)FK(curcumin)SESY - Formula (XXXV) thatcomprises the peptide compound having SEQ ID NO: 15 wherein each lysineresidue has a curcumin molecule connected thereto Cabazitaxel-conjugatesKBD-105 (2:1) Acetyl-GVRAK(cabazitaxel)AGVRNMFK(cabazitaxel)SESY -Formula (XXXI) that comprises the peptide compound having SEQ ID NO: 14wherein each lysine residue has a cabazitaxel molecule connected theretoKBD-106 (2:1) Acetyl-GVRAK(cabazitaxel)AGVRN(Nle)FK(cabazitaxel)SESY -Formula (XXXII) that comprises the peptide compound having SEQ ID NO: 15wherein each lysine residue has a cabazitaxel molecule connected theretoKBD-201 (2:1)Acetyl-YK(cabazitaxel)SLRRK(cabazitaxel)APRWDAPLRDPALRQLL - Formula(XXXIII) that comprises the peptide compound having SEQ ID NO: 16wherein each lysine residue has a cabazitaxel molecule connected thereto

The term “conjugating” au used herein, refers, for example, to thepreparation of a conjugate as defined above. Such an action comprisesconnecting a peptide compound together with at least one therapeuticagent, optionally via a linker.

For example, the following are general chemical formulas of someconjugate compounds herein disclosed.

Docetaxel-Katana Peptide Conjugate:

Doxorubicin-Katana Peptide Conjugate:

Curcumin-Katana Peptide Conjugate:

For example, the following are the chemical structures of some conjugatecompounds herein disclosed.

Docetaxel-KBP-102 Drug (3:1) or KBA-102:

Docetaxel-KBP105 Drug (2:1) or KBA-105:

Curcumin-KBP106 (2:1) or KBC-106:

The term “linker” as used herein means a chemical structure connecting apeptide compound herein disclosed to at least one therapeutic agent. Thelinker can be connected to the peptide compound at different functionalgroups on the peptide compounds. For example, the linker can beconnected to the peptide compound at the primary amines (amines (—NH2):this group exists at the N-terminus of each polypeptide chain (calledthe alpha-amine) and in the side chain of lysine (Lys, K) residues(called the epsilon-amine). For example, the linker can be connected tothe peptide compound at the carboxyls (—COOH): this group exists at theC-terminus of each polypeptide chain and in the side chains of asparticacid (Asp, D) and glutamic acid (Glu, E). For example, the linker can beconnected to the peptide compound at the Sulfhydryls (—SH): This groupexists in the side chain of cysteine (Cys, C). Often, as part of aprotein's secondary or tertiary structure, cysteines are joined togetherbetween their side chains via disulfide bonds (—S—S—). These must bereduced to sulfhydryls to make them available for crosslinking by mosttypes of reactive groups. For example, the linker can be connected tothe peptide compound at the Carbonyls (—CHO): Ketone or aldehyde groupscan be created in glycoproteins by oxidizing the polysaccharidepost-translational modifications (glycosylation) with sodiummeta-periodate. For example, the linker can be a cleavable linker. Forexample, the linker can be a non-cleavable linker.

The following table summarizes the reactivity class and the chemicalgroup of some of the principals linkers for standard chemicalconjugation:

Reactivity class Chemical group Carboxyl-to-amine reactive groupsCarbodiimide (e.g., EDC) Amine-reactive groups NHS ester ImidoesterPentafluorophenyl ester Hydroxymethyl phosphine Sulfhydryl-reactivegroups Maleimide Haloacetyl (Bromo- or Iodo-) PyridyldisulfideThiosulfonate Vinylsulfone Aldehyde-reactive groups Hydrazide i.e.,oxidized sugars (carbonyls) Alkoxyamine Photoreactive groups DiazirineAryl Azide

For example, homobifunctional and heterobifunctional crosslinkers can beused. For example, Disuccinimidyl suberate (DSS) is a homobifunctionalcrosslinker that has identical amine-reactive NHS-ester groups at eitherend of a short spacer arm. For example, Sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC) is aheterobifunctional crosslinker that has an amine-reactivesulfo-NHS-ester group at one end and a sulfhydryl reactive maleimidegroup at the opposite end of a cyclohexane spacer arm. This allows forsequential, two-step conjugation procedures. Among the commerciallyavailable homobifunctional cross-linkers are: BSOCOES(Bis(2-[Succinimidooxycarbonyloxy]ethyl) sulfone; DPDPB(1,4-Di-(3′-[2pyridyldithio]-propionamido) butane; DSS (disuccinimidylsuberate); DST (disuccinimidyl tartrate); Sulfo DST (sulfodisuccinimidyltartrate); DSP (dithiobis(succinimidyl propionate); DTSSP(3,3′-Dithiobis(sulfosuccinimidyl propionate); EGS (ethylene glycolbis(succinimidyl succinate)); and BASED(Bis(p-[4-azidosalicylamido]-ethyl)disulfide iodinatable).

The polypeptides may be conjugated through a variety of linkers, e.g.,sulfhydryl groups, amino groups (amines), or any appropriate reactivegroup. The linker can be a covalent bond. The linker group may comprisea flexible arm, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15carbon atoms.

Exemplary linkers include, without limitation, pyridinedisulfide,thiosulfonate, vinylsulfonate, isocyanate, imidoester, diazine,hydrazine, thiol, carboxylic acid, multi-peptide linkers, and acetylene.Alternatively other linkers that can be used include BS³[Bis(sulfosuccinimidyl)suberate] (which is a homobifunctionalN-hydroxysuccinimide ester that targets accessible primary amines),NHS/EDC (N-hydroxysuccinimide and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS/EDC allows for theconjugation of primary amine groups with carboxyl groups), sulfo-EMCS([N-ε-maleimidocaproic acid]hydrazide (sulfo-EMCS are heterobifunctionalreactive groups that are reactive toward sulfhydryl and amino groups),hydrazide (most proteins contain exposed carbohydrates and hydrazide isa useful reagent for linking carboxyl groups to primary amines).

To form covalent bonds, one can use as a chemically reactive group awide variety of active carboxyl groups (e.g., esters) where the hydroxylmoiety is physiologically acceptable at the levels required to modifythe peptide. Particular agents include for example N-hydroxysuccinimide(NHS), N-hydroxy-sulfosuccinimide (sulfo-NHS),maleimide-benzoyl-succinimide (MBS), gamma-maleimido-butyryloxysuccinimide ester (GMBS), maleimido propionic acid (MPA), maleimidohexanoic acid (MHA), and maleimido undecanoic acid (MUA).

Primary amines are the principal targets for NHS esters; NHS estersreact with primary amines to form covalent amide bonds. Accessiblea-amine groups present on the N-termini of proteins and the ε-amine oflysine react with NHS esters. Thus, conjugated compounds hereindisclosed can include a linker having a NHS ester conjugated to anN-terminal amino of a peptide or to an ε-amine of lysine. An amide bondis formed when the NHS ester reacts with primary amines releasingN-hydroxysuccinimide. Succinimide containing reactive groups may bereferred to more simply as succinimidyl groups. In some embodiments, thefunctional group on the protein will be a thiol group and the chemicallyreactive group will be a maleimido-containing group such asgamma-maleimide-butylamide (GMBA or MPA). Such maleimide-containinggroups may be referred to herein as maleido groups.

Amine-to-amine linkers include NHS esters, imidoesters, and others,examples of which are listed below.

Exemplary NHS esters: DSG (disuccinimidyl glutarate) DSS (disuccinimidylsuberate) BS³ (bis[sulfosuccinimidyl] suberate) TSAT (tris-succinimidylaminotriacetate) Variants of bis-succinimide ester-activated compoundsincluding a polyethylene glycol spacer such as BS(PEG)_(n) where n is1-20 (e.g., BS(PEG)₅ and BS(PEG)₉) DSP (Dithiobis[succinimidylpropionate]) DTSSP (3,3′-dithiobis[sulfosuccinimidylpropionate]) DST(disuccinimidyl tartarate) BSOCOES(bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone) EGS (ethylene glycolbis[succinimidylsuccinate]) sulfo-EGS (ethylene glycolbis[sulfosuccinimidylsuccinate]) Exemplary imidoesters: DMA (dimethyladipimidate•2 HCl) DMP (dimethyl pimelimidate•2 HCl) DMS (dimethylsuberimidate•2 HCl) DTBP (dimethyl 3,3′-dithiobispropionimidate•2 HCl)Other exemplary amine-to-amine linkers: DFDNB(1,5-difluoro-2,4-dinitrobenzene) THPP (β-[tris(hydroxymethyl)phosphino] propionic acid (betaine))

The linker may also be a sulfhydryl-to-sulfhydryl linker, such as themaleimides and pyridyldithiols listed below.

Exemplary maleimides: Another sulfhydryl linker: BMOE(bis-maleimidoethane) HBVS (1,6-hexane-bis-vinylsulfone) BMB(1,4-bismaleimidobutane) BMH (bismaleimidohexane) TMEA(tris[2-maleimidoethyl]amine) BM(PEG)21,8-bis-maleimidodiethyleneglycol) BM(PEG)_(n,) where n is 1 to 20(e.g., 2 or 3) BMDB (1,4 bismaleimidyl-2,3-dihydroxybutane) DTME(dithio-bismaleimidoethane) Exemplary pyridyldithiol: DPDPB(1,4-di-[3′-(2′-pyridyldithio)-propionamido]butane)

The linker may be an amine-to-sulfhydryl linker, which includes NHSester/maleimide compounds. Examples of these compounds are providedbelow.

Amine-to-sulfhydryl linkers: AMAS (N-(α-maleimidoacetoxy)succinimideester) BMPS (N-[β-maleimidopropyloxy]succinimide ester) GMBS(N-[γ-maleimidobutyryloxy]succinimide ester) sulfo-GMBS(N-[γ-maleimidobutyryloxy]sulfosuccinimide ester) MBS(m-maleimidobenzoyl-N-hydroxysuccinimide ester) sulfo-MBS(m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester) SMCC (succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate) sulfo-SMCC(Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate) EMCS([N-ε-maleimidocaproyloxy]succinimide ester) Sulfo-EMCS([N-ε-maleimidocaproyloxy]sulfosuccinimide ester) SMPB (succinimidyl4-[p-maleimidophenyl]butyrate) sulfo-SMPB (sulfosuccinimidyl4-[p-maleimidophenyl]butyrate) SMPH(succinimidyl-6-[β-maleimidopropionamido]hexanoate) LC-SMCC(succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate])sulfo-KMUS (N-[κ-maleimidoundecanoyloxy]sulfosuccinimide ester)SM(PEG)_(n) (succinimidyl-([N-maleimidopropionamido-polyethyleneglycol)ester), where n is 1 to 30 (e.g., 2, 4, 6, 8, 12, or 24) SPDP(N-succinimidyl 3-(2-pyridyldithio)-propionate) LC-SPDP (succinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate) sulfo-LC-SPDP(sulfosuccinimidyl 6-(3′-[2-pyridyldithio]-propionamido)hexanoate) SMPT(4-succinimidyloxycarbonyl-α-methyl-α-[2-pyridyldithio]toluene)Sulfo-LC-SMPT(4-sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate)SIA (N-succinimidyl iodoacetate) SBAP (succinimidyl3-[bromoacetamido]propionate) SIAB(N-succinimidyl[4-iodoacetyl]aminobenzoate) sulfo-SIAB(N-sulfosuccinimidyl[4-iodoacetyl]aminobenzoate)

The linker can react with an amino group and a non-selective entity.Such linkers include NHS ester/aryl azide and NHS ester/diazirinelinkers, examples of which are listed below.

NHS ester/aryl azide linkers: NHS-ASA(N-hydroxysuccinimidyl-4-azidosalicylic acid) ANB-NOS(N-5-azido-2-nitrobenzoyloxysuccinimide) sulfo-HSAB(N-hydroxysulfosuccinimidyl-4-azidobenzoate) sulfo-NHS-LC-ASA(sulfosuccinimidyl[4-azidosalicylamido]hexanoate) SANPAH(N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate) sulfo-SANPAH(N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate)sulfo-SFAD(sulfosuccinimidyl-(perfluoroazidobenzamido)-ethyl-1,3′-dithioproprionate)sulfo-SAND(sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-proprionate)sulfo-SAED (sulfosuccinimidyl2-[7-amino-4-methylcoumarin-3-acetamido]ethyl- 1,3′dithiopropionate) NHSester/diazirine linkers: SDA (succinimidyl 4,4′-azipentanoate) LC-SDA(succinimidyl 6-(4,4′-azipentanamido)hexanoate) SDAD (succinimidyl2-([4,4′-azipentanamido]ethyl)-1,3′-dithioproprionate) sulfo-SDA(sulfosuccinimidyl 4,4′-azipentanoate) sulfo-LC-SDA (sulfosuccinimidyl6-(4,4′-azipentanamido)hexanoate) sulfo-SDAD (sulfosuccinimidyl2-([4,4′-azipentanamido]ethyl)-1,3′-dithioproprionate)

Exemplary amine-to-carboxyl linkers include carbodiimide compounds(e.g., DCC (N,N-dicyclohexylcarbodimide) and EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide)). Exemplarysulfhydryl-to-nonselective linkers include pyridyldithiol/aryl azidecompounds (e.g., APDP((N-[4-(p-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide)).Exemplary sulfhydryl-to-carbohydrate linkers include maleimide/hydrazidecompounds (e.g., BMPH (N-[β-maleimidopropionic acid]hydrazide), EMCH([N-ε-maleimidocaproic acid]hydrazide), MPBH4-(4-N-maleimidophenyl)butyric acid hydrazide), and KMUH(N-[κ-maleimidoundecanoic acid]hydrazide)) and pyridyldithiol/hydrazidecompounds (e.g., PDPH (3-(2-pyridyldithio)propionyl hydrazide)).Exemplary carbohydrate-to-nonselective linkers include hydrazide/arylazide compounds (e.g., ABH (p-azidobenzoyl hydrazide)). Exemplaryhydroxyl-to-sulfhydryl linkers include isocyanate/maleimide compounds(e.g., (N-[p-maleimidophenyl]isocyanate)). Exemplary amine-to-DNAlinkers include NHS ester/psoralen compounds (e.g., SPB(succinimidyl-[4-(psoralen-8-yloxy)]-butyrate)).

To generate a branch point of varying complexity in a conjugate peptide,the linker can be capable of linking 3-7 entities.

Exemplary tri-functional linkers:

LC-TSAT (tris-succinimidyl (6- aminocaproyl)aminotriacetate), trist-succinimidyl-1,3,5-benzenetricarboxylate MDSI(maleimido-3,5-disuccinimidyl isophthalate)

SDMB (succinimidyl-3,5- dimaleimidophenyl benzoate Mal-4(tetrakis-(3-maleimidopropyl) pentaerythritol, NHS-4 (tetrakis-(N-succinimidylcarboxypropyl)pentaerythritol))

TMEA and TSAT reach through their maleimide groups with sulfhydrylgroups. The hydroxyl groups and carboxy group of THPP can react withprimary or secondary amines. Other useful linkers conform to the formulaY═C═N-Q-A-C(O)—Z, where Q is a homoaromatic or heteroaromatic ringsystem; A is a single bond or an unsubstituted or substituted divalentC₁₋₃₀ bridging group, Y is O or S; and Z is Cl, Br, I, N₃,N-succinimidyloxy, imidazolyl, 1-benzotriazolyloxy, OAr where Ar is anelectron-deficient activating aryl group, or OC(O)R where R is-A-Q-N═C═Y or C₄-20 tertiary-alkyl (see U.S. Pat. No. 4,680,338).

Other useful linkers have the formula

where R₁ is H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₆₋₁₂ aryl or aralkyl or thesecoupled with a divalent organic —O—, —S—, or

where R′ is C₁₋₆ alkyl, linking moiety; R₂ is H, C₁₋₁₂ alkyl, C₆₋₁₂aryl, or C₆₋₁₂ aralkyl, R₃ is

or another chemical structure that is able to delocalize the lone pairelectrons of the adjacent nitrogen and R₄ is a pendant reactive groupcapable of linking R₃ to a peptide vector or to an agent (see forexample U.S. Pat. No. 5,306,809).

The linker may include at least one amino acid residue and can be apeptide of at least or about 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30, 40,or 50 amino acid residues. Where the linker is a single amino acidresidue it can be any naturally or non-naturally occurring amino acid(e.g., Gly or Cys). Where the linker is a short peptide, it can be aglycine-rich peptide (which tend to be flexible) such as a peptidehaving the sequence [Gly-Gly-Gly-Gly-Ser]_(n) where n is an integer from1 to 6, inclusive (see U.S. Pat. No. 7,271,149) or a serine-rich peptidelinker (see U.S. Pat. No. 5,525,491). Serine rich peptide linkersinclude those of the formula [X—X—X—X-Gly], where up to two of the X areThr, the remaining X are Ser, and y is an integer from 1 to 5, inclusive(e.g., Ser-Ser-Ser-Ser-Gly, where y is greater than 1). Other linkersinclude rigid linkers (e.g., PAPAP and (PT)_(n)P, where n is 2, 3, 4, 5,6, or 7) and a-helical linkers (e.g., A(EAAAK)_(n)A, where n is 1, 2, 3,4, or 5).

The linker can be an aliphatic linker (e.g., with an amide bond to thepolypeptide and an ester bond to the therapeutic agent). Where analiphatic linker is used, it may vary with regard to length (e.g.C₁-C₂₀) and the chemical moieties it includes (e.g., an amino group orcarbamate).

Examples of suitable amino acid linkers are succinic acid, Lys, Glu, andAsp, or a dipeptide such as Gly-Lys. When the linker is succinic acid,one carboxyl group thereof may form an amide bond with an amino group ofthe amino acid residue, and the other carboxyl group thereof may, forexample, form an amide bond with an amino group of the peptide orsubstituent. When the linker is Lys, Glu, or Asp, the carboxyl groupthereof may form an amide bond with an amino group of the amino acidresidue, and the amino group thereof may, for example, form an amidebond with a carboxyl group of the substituent. When Lys is used as thelinker, a further linker may be inserted between the ε-amino group ofLys and the substituent. The further linker may be succinic acid, whichcan form an amide bond with the ε-amino group of Lys and with an aminogroup present in the substituent. In one embodiment, the further linkeris Glu or Asp (e.g., which forms an amide bond with the ε-amino group ofLys and another amide bond with a carboxyl group present in thesubstituent), that is, the substituent is a NE-acylated lysine residue.

The linker can also be a branched polypeptide. Exemplary branchedpeptide linkers are described in U.S. Pat. No. 6,759,509.

The linker can provide a cleavable linkage (e.g., a thioester linkage)or a non-cleavable linkage (e.g., a maleimide linkage). For example, acytotoxic protein can be bound to a linker that reacts with modifiedfree amines, which are present at lysine residues within the polypeptideand at the amino-terminus of the polypeptide. Thus, linkers useful inthe present conjugate compounds can comprise a group that is reactivewith a primary amine on the polypeptide or modified polypeptide to whichthe therapeutic agent moiety is conjugated. More specifically, thelinker can be selected from the group consisting of monofluorocyclooctyne (MFCO), bicyclo[6.1.0]nonyne (BCN),N-succinimidyl-S-acetylthioacetate (SATA),N-succinimidyl-S-acetylthiopropionate (SATP), maleimido anddibenzocyclooctyne ester (a DBCO ester). Useful cyclooctynes, within agiven linker, include OCT, ALO, MOFO, DIFO, DIBO, BARAC, DIBAC, andDIMAC.

The linker may comprise a flexible arm, such as for example, a short arm(<2 carbon chain), a medium-size arm (from 2-5 carbon chain), or a longarm (3-6 carbon chain).

Click chemistry can also be used for conjugation on a peptide (DBCO,TCO, tetrazine, azide and alkyne linkers). These families of linkers canbe reactive toward amine, carboxyl and sulfhydryl groups. In addition,these linkers can also be biotinylated, pegylated, modified with afluorescent imaging dye, or phosphoramidited for incorporation onto anoligonucleotide sequence.

The term “intermediate” as used herein refers to a therapeutic agentthat has been reacted with a linker thereby forming an intermediate oran activated form of the therapeutic agent. The intermediate can bereacted with a peptide compound herein disclosed thereby forming aconjugate compound herein disclosed that can be used for treating acancer.

The term “amino acid” refers to the common natural (genetically encoded)or synthetic amino acids and common derivatives thereof, known to thoseskilled in the art. When applied to amino acids, “standard” or“proteinogenic” refers to the genetically encoded 20 amino acids intheir natural configuration. Similarly, when applied to amino acids,“non-standard,” “unnatural” or “unusual” refers to the wide selection ofnon-natural, rare or synthetic amino acids such as those described byHunt, S. in Chemistry and Biochemistry of the Amino Acids, Barrett, G.C., ed., Chapman and Hall: New York, 1985. Some examples of non-standardamino acids include non-alpha amino acids, D-amino acids.

Abbreviations used for amino acids and designation of peptides followthe rules of the IUPAC-IUB Commission of Biochemical Nomenclature in J.Biol. Chem. 1972, 247, 977-983. This document has been updated: Biochem.J., 1984, 219, 345-373; Eur. J. Biochem., 1984, 138, 9-37; 1985, 152, 1;Int. J. Pept. Prot. Res., 1984, 24, following p 84; J. Biol. Chem.,1985, 260, 14-42; Pure Appl. Chem. 1984, 56, 595-624; Amino Acids andPeptides, 1985, 16, 387-410; and in Biochemical Nomenclature and RelatedDocuments, 2^(nd) edition, Portland Press, 1992, pp 39-67. Extensions tothe rules were published in the JCBN/NC-IUB Newsletter 1985, 1986, 1989;see Biochemical Nomenclature and Related Documents, 2^(nd) edition,Portland Press, 1992, pp 68-69.

The term “antagonist” refers to a compound that reduces at least some ofthe effect of the endogenous ligand of a protein, receptor, enzyme,interaction, or the like.

The term “inhibitor” refers to a compound that reduces the normalactivity of a protein, receptor, enzyme, interaction, or the like.

The term “inverse agonist” refers to a compound that reduces theactivity of a constitutively-active receptor below its basal level.

The term “library” refers to a collection of compounds that can be usedfor example for drug discovery purposes. For example, the librarycompounds can be peptide compounds and/or conjugate compounds hereindisclosed.

The term “mixture” as used herein, means a composition comprising two ormore compounds. In an embodiment a mixture is a mixture of two or moredistinct compounds. In a further embodiment, when a compound is referredto as a “mixture”, this means that it can comprise two or more “forms”of the compounds, such as, salts, solvates, prodrugs or, whereapplicable, stereoisomers of the compound in any ratio. A person ofskill in the art would understand that a compound in a mixture can alsoexist as a mixture of forms. For example, a compound may exist as ahydrate of a salt or as a hydrate of a salt of a prodrug of thecompound. All forms of the compounds disclosed herein are within thescope of the present application.

The term “modulator” refers to a compound that imparts an effect on abiological or chemical process or mechanism. For example, a modulatormay increase, facilitate, upregulate, activate, inhibit, decrease,block, prevent, delay, desensitize, deactivate, down regulate, or thelike, a biological or chemical process or mechanism. Accordingly, amodulator can be an “agonist” or an “antagonist.” Exemplary biologicalprocesses or mechanisms affected by a modulator include, but are notlimited to, enzyme binding, receptor binding and hormone release orsecretion. Exemplary chemical processes or mechanisms affected by amodulator include, but are not limited to, catalysis and hydrolysis.

The term “peptide” refers to a chemical compound comprising at least twoamino acids covalently bonded together using amide bonds.

The term “prodrug” as used herein refers to a derivative of an activeform of a known compound or composition which derivative, whenadministered to a subject, is gradually converted to the active form toproduce a better therapeutic response and/or a reduced toxicity level.In general, prodrugs will be functional derivatives of the compoundsdisclosed herein which are readily convertible in vivo into the compoundfrom which it is notionally derived. Prodrugs include, withoutlimitation, acyl esters, carbonates, phosphates, and urethanes. Thesegroups are exemplary and not exhaustive, and one skilled in the artcould prepare other known varieties of prodrugs. Prodrugs may be, forexample, formed with available hydroxy, thiol, amino or carboxyl groups.For example, the available OH and/or NH₂ in the compounds of thedisclosure may be acylated using an activated acid in the presence of abase, and optionally, in inert solvent (e.g. an acid chloride inpyridine). Some common esters which have been utilized as prodrugs arephenyl esters, aliphatic (C₁-C₂₄) esters, acyloxymethyl esters,carbamates and amino acid esters. In certain instances, the prodrugs ofthe compounds of the disclosure are those in which the hydroxy and/oramino groups in the compounds is masked as groups which can be convertedto hydroxy and/or amino groups in vivo. Conventional procedures for theselection and preparation of suitable prodrugs are described, forexample, in “Design of Prodrugs” ed. H. Bundgaard, Elsevier, 1985.

The term “protecting group” refers to any chemical compound that may beused to prevent a potentially reactive functional group, such as anamine, a hydroxyl or a carboxyl, on a molecule from undergoing achemical reaction while chemical change occurs elsewhere in themolecule. A number of such protecting groups are known to those skilledin the art and examples can be found in Protective Groups in OrganicSynthesis, T. W. Greene and P. G. Wuts, eds., John Wiley & Sons, NewYork, 4^(th) edition, 2006, 1082 pp, ISBN 9780471697541. Examples ofamino protecting groups include, but are not limited to, phthalimido,trichloroacetyl, benzyloxycarbonyl, tert butoxycarbonyl, andadamantyl-oxycarbonyl. In some embodiments, amino protecting groups arecarbamate amino protecting groups, which are defined as an aminoprotecting group that when bound to an amino group forms a carbamate. Inother embodiments, amino carbamate protecting groups areallyloxycarbonyl (Alloc), benzyloxycarbonyl (Cbz), 9fluorenylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc) and α,αdimethyl-3,5 dimethoxybenzyloxycarbonyl (Ddz). For a recent discussionof newer nitrogen protecting groups see: Tetrahedron 2000, 56,2339-2358. Examples of hydroxyl protecting groups include, but are notlimited to, acetyl, tert-butyldimethylsilyl (TBDMS), trityl (Trt),tert-butyl, and tetrahydropyranyl (THP). Examples of carboxyl protectinggroups include, but are not limited to, methyl ester, tert-butyl ester,benzyl ester, trimethylsilylethyl ester, and 2,2,2-trichloroethyl ester.

The term “sequence identity” as used herein refers to the percentage ofsequence identity between two polypeptide sequences or two nucleic acidsequences. To determine the percent identity of two amino acid sequencesor of two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino acid or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical overlappingpositions/total number of positions.times.100%). In one embodiment, thetwo sequences are the same length. The determination of percent identitybetween two sequences can also be accomplished using a mathematicalalgorithm. A preferred, non-limiting example of a mathematical algorithmutilized for the comparison of two sequences is the algorithm of Karlinand Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modifiedas in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A.90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLASTnucleotide searches can be performed with the NBLAST nucleotide programparameters set, e.g., for score=100, wordlength=12 to obtain nucleotidesequences homologous to a nucleic acid molecules of the presentapplication. BLAST protein searches can be performed with the XBLASTprogram parameters set, e.g., to score-50, wordlength=3 to obtain aminoacid sequences homologous to a protein molecule of the presentdisclosure. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., 1997, NucleicAcids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to performan iterated search which detects distant relationships between molecules(Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, thedefault parameters of the respective programs (e.g., of XBLAST andNBLAST) can be used (see, e.g., the NCBI website). Another preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, 1988,CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. The percent identity between twosequences can be determined using techniques similar to those describedabove, with or without allowing gaps. In calculating percent identity,typically only exact matches are counted.

The expression “consisting essentially of”, as used herein, is intendedto specify the presence of the stated features, elements, components,groups, integers, and/or steps as well as those that do not materiallyaffect the basic and novel characteristic(s) of features, elements,components, groups, integers, and/or steps.

The term “solid phase chemistry” refers to the conduct of chemicalreactions where one component of the reaction is covalently bonded to apolymeric material (solid support as defined below). Reaction methodsfor performing chemistry on solid phase have become more widely knownand established outside the traditional fields of peptide andoligonucleotide chemistry (Solid-Phase Synthesis: A Practical Guide, F.Albericio, ed., CRC Press, 2000, 848 pp, ISBN: 978-0824703592; OrganicSynthesis on Solid Phase, 2^(nd) edition, Florencio Zaragoza Dörwald,Wiley-VCH, 2002, 530 pp, ISBN: 3-527-30603-X; Solid-Phase OrganicSynthesis: Concepts, Strategies, and Applications, P. H. Toy, Y. Lam,eds., Wiley, 2012, 568 pp, ISBN: 978-0470599143).

The term “solid support,” “solid phase” or “resin” refers to amechanically and chemically stable polymeric matrix utilized to conductsolid phase chemistry. This is denoted by “Resin,” “P—” or the followingsymbol:

.

Examples of appropriate polymer materials include, but are not limitedto, polystyrene, polyethylene, polyethylene glycol (PEG, including, butnot limited to, ChemMatrix® (Matrix Innovation, Quebec, Quebec, Canada;J. Comb. Chem. 2006, 8, 213-220)), polyethylene glycol grafted orcovalently bonded to polystyrene (also termed PEG-polystyrene,TentaGel™, Rapp, W.; Zhang, L.; Bayer, E. In Innovations andPerspectives in Solid Phase Synthesis. Peptides, Polypeptides andOligonucleotides; Epton, R., ed.; SPCC Ltd.: Birmingham, UK; p 205),polyacrylate (CLEAR™) polyacrylamide, polyurethane, PEGA[polyethyleneglycol poly(N,N dimethyl-acrylamide) co-polymer,Tetrahedron Lett. 1992, 33, 3077-3080], cellulose, etc. These materialscan optionally contain additional chemical agents to form cross-linkedbonds to mechanically stabilize the structure, for example polystyrenecross-linked with divinylbenezene (DVB, usually 0.1-5%, preferably0.5-2%). This solid support can include as non-limiting examplesaminomethyl polystyrene, hydroxymethyl polystyrene, benzhydrylaminepolystyrene (BHA), methylbenzhydrylamine (MBHA) polystyrene, and otherpolymeric backbones containing free chemical functional groups, mosttypically, NH₂ or —OH, for further derivatization or reaction. The termis also meant to include “Ultraresins” with a high proportion(“loading”) of these functional groups such as those prepared frompolyethyleneimines and cross-linking molecules (J. Comb. Chem. 2004, 6,340-349). At the conclusion of the synthesis, resins are typicallydiscarded, although they have been shown to be able to be recycled(Tetrahedron Lett. 1975, 16, 3055).

In general, the materials used as resins are insoluble polymers, butcertain polymers have differential solubility depending on solvent andcan also be employed for solid phase chemistry. For example,polyethylene glycol can be utilized in this manner since it is solublein many organic solvents in which chemical reactions can be conducted,but it is insoluble in others, such as diethyl ether. Hence, reactionscan be conducted homogeneously in solution, then the product on thepolymer precipitated through the addition of diethyl ether and processedas a solid. This has been termed “liquid-phase” chemistry.

The expression “pharmaceutically acceptable” means compatible with thetreatment of subjects such as animals or humans.

The expression “pharmaceutically acceptable salt” means an acid additionsalt or basic addition salt which is suitable for or compatible with thetreatment of subjects such as animals or humans.

The expression “pharmaceutically acceptable acid addition salt” as usedherein means any non-toxic organic or inorganic salt of any compound ofthe present disclosure, or any of its intermediates. Illustrativeinorganic acids which form suitable salts include hydrochloric,hydrobromic, sulfuric and phosphoric acids, as well as metal salts suchas sodium monohydrogen orthophosphate and potassium hydrogen sulfate.Illustrative organic acids that form suitable salts include mono-, di-,and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic,succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic,benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonicacids such as p-toluenesulfonic and methanesulfonic acids. Either themono or di-acid salts can be formed, and such salts may exist in eithera hydrated, solvated or substantially anhydrous form. In general, theacid addition salts of the compounds of the present disclosure are moresoluble in water and various hydrophilic organic solvents, and generallydemonstrate higher melting points in comparison to their free baseforms. The selection of the appropriate salt will be known to oneskilled in the art. Other non-pharmaceutically acceptable salts, e.g.oxalates, may be used, for example, in the isolation of the compounds ofthe present disclosure, for laboratory use, or for subsequent conversionto a pharmaceutically acceptable acid addition salt.

The term “pharmaceutically acceptable basic addition salt” as usedherein means any non-toxic organic or inorganic base addition salt ofany acid compound of the disclosure, or any of its intermediates. Acidiccompounds of the disclosure that may form a basic addition salt include,for example, where CO₂H is a functional group. Illustrative inorganicbases which form suitable salts include lithium, sodium, potassium,calcium, magnesium or barium hydroxide. Illustrative organic bases whichform suitable salts include aliphatic, alicyclic or aromatic organicamines such as methylamine, trimethylamine and picoline or ammonia. Theselection of the appropriate salt will be known to a person skilled inthe art. Other non-pharmaceutically acceptable basic addition salts, maybe used, for example, in the isolation of the compounds of thedisclosure, for laboratory use, or for subsequent conversion to apharmaceutically acceptable acid addition salt.

The formation of a desired compound salt is achieved using standardtechniques. For example, the neutral compound is treated with an acid orbase in a suitable solvent and the formed salt is isolated byfiltration, extraction or any other suitable method.

The formation of a desired compound salt is achieved using standardtechniques. For example, the neutral compound is treated with an acid orbase in a suitable solvent and the formed salt is isolated byfiltration, extraction or any other suitable method.

The term “solvate” as used herein means a compound or itspharmaceutically acceptable salt, wherein molecules of a suitablesolvent are incorporated in the crystal lattice. A suitable solvent isphysiologically tolerable at the dosage administered. Examples ofsuitable solvents are ethanol, water and the like. When water is thesolvent, the molecule is referred to as a “hydrate”. The formation ofsolvates will vary depending on the compound and the solvate. Ingeneral, solvates are formed by dissolving the compound in theappropriate solvent and isolating the solvate by cooling or using anantisolvent. The solvate is typically dried or azeotroped under ambientconditions.

The term “subject” as used herein includes all members of the animalkingdom including mammals such as a mouse, a rat, a dog and a human.

The terms “suitable” and “appropriate” mean that the selection of theparticular group or conditions would depend on the specific syntheticmanipulation to be performed and the identity of the molecule but theselection would be well within the skill of a person trained in the art.All process steps described herein are to be conducted under conditionssuitable to provide the product shown. A person skilled in the art wouldunderstand that all reaction conditions, including, for example,reaction solvent, reaction time, reaction temperature, reactionpressure, reactant ratio and whether or not the reaction should beperformed under an anhydrous or inert atmosphere, can be varied tooptimize the yield of the desired product and it is within their skillto do so.

The expression a “therapeutically effective amount”, “effective amount”or a “sufficient amount” of a compound or composition of the presentdisclosure is a quantity sufficient to, when administered to thesubject, including a mammal, for example a human, effect beneficial ordesired results, including clinical results, and, as such, a“therapeutically effective amount” or an “effective amount” depends uponthe context in which it is being applied. For example, in the context oftreating cancer, it is an amount of the compound or compositionsufficient to achieve such treatment of the cancer as compared to theresponse obtained without administration of the compound or composition.The amount of a given compound or composition of the present disclosurethat will correspond to an effective amount will vary depending uponvarious factors, such as the given drug or compound, the pharmaceuticalformulation, the route of administration, the type of disease ordisorder, the identity of the subject or host being treated, and thelike, but can nevertheless be routinely determined by one skilled in theart. Also, as used herein, a “therapeutically effective amount” or“effective amount” of a compound or composition of the presentdisclosure is an amount which inhibits, suppresses or reduces a cancer(e.g., as determined by clinical symptoms or the amount of cancerouscells) in a subject as compared to a control.

As used herein, and as well understood in the art, “treatment” or“treating” is an approach for obtaining beneficial or desired results,including clinical results. Beneficial or desired clinical results caninclude, but are not limited to, decrease in tumour progression,decrease in tumour size, decrease in tumour growth rate, decrease intumor invasion and metastatic potential, alleviation or amelioration ofone or more symptoms or conditions, diminishment of extent of disease,stabilized (i.e. not worsening) state of disease, preventing spread ofdisease, delay or slowing of disease progression, amelioration orpalliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” or “treating”can also mean prolonging survival as compared to expected survival ifnot receiving treatment.

The term “tolerability” or “tolerated” as used herein means a degree towhich a therapeutic agent may be endured or accepted by a subjecttreated with the therapeutic agent. For example, tolerability may beassessed by measuring different parameters such as (i) maintaining orabsence of weight loss, (ii) duration of treatment withstood and (iii)decrease or absence of side effects. For example, it is well establishedthat a therapeutic agent is tolerated by a subject when there is noweight loss observed during treatment using such a therapeutic agent.

The term “administered” or “administering” as used herein meansadministration of a therapeutically effective amount of a compound orcomposition of the application to a cell either in vitro (e.g. a cellculture) or in vivo (e.g. in a subject).

In understanding the scope of the present disclosure, the term“comprising” and its derivatives, as used herein, are intended to beopen ended terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but do not excludethe presence of other unstated features, elements, components, groups,integers and/or steps. The foregoing also applies to words havingsimilar meanings such as the terms, “including”, “having” and theirderivatives. Finally, terms of degree such as “substantially”, “about”and “approximately” as used herein mean a reasonable amount of deviationof the modified term such that the end result is not significantlychanged. These terms of degree should be construed as including adeviation of at least ±5% of the modified term if this deviation wouldnot negate the meaning of the word it modifies.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural references unless the contentclearly dictates otherwise. Thus for example, a composition containing“a compound” includes a mixture of two or more compounds. It should alsobe noted that the term “or” is generally employed in its sense including“and/or” unless the content clearly dictates otherwise.

In compositions comprising an “additional” or “second” component, thesecond component as used herein is chemically different from the othercomponents or first component. A “third” component is different from theother, first, and second components, and further enumerated or“additional” components are similarly different.

The definitions and embodiments described in particular sections areintended to be applicable to other embodiments herein described forwhich they are suitable as would be understood by a person skilled inthe art.

The recitation of numerical ranges by endpoints herein includes allnumbers and fractions subsumed within that range (e.g. 1 to 5 includes1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood thatall numbers and fractions thereof are presumed to be modified by theterm “about.”

A platform allowing the transport of therapeutic agents into cancercells for new therapies directed against primary and secondary tumourshas recently been developed. This approach utilizes peptide compoundsderived from bacterial proteins or from ligands of receptors expressedin cancer cells (ex. sortilins/syndecans). In the present disclosure,the conjugation of anticancer agents and phytochemicals to one of thesepeptide compounds is described. For example, anticancer agents, forexample Docetaxel, Cabazitaxel and Doxorubicin, can be conjugated to thepeptide compounds. Phytochemicals, for example curcumin, can also beconjugated to the peptide compounds. Moreover, after conjugation toKatana peptide, uptake of the conjugated Katana peptide is unaffected bythe P-gp inhibitor Cyclosporine A, confirming that Katana-peptides arenot substrates for efflux pumps such as P-gp. These results furtherindicate that these peptide-drug conjugates could be used in otherapplications outside of oncology. In addition to inducing greater tumourapoptosis compared to unconjugated therapeutic agent, the conjugatecompounds herein described may also provide greater tolerability andreduced toxicity.

Disclosed herein are peptide compounds as well as conjugate compoundscomprising at least one therapeutic agent connected to a peptidecompound. Such compounds can be used for the treatment of cancer, forexample a cancer involving sortilin expression.

Accordingly, a first aspect is a peptide compound having at least 80%sequence identity to a compound chosen from compounds of formula (I),formula (II), formula (III), formula (IV), formula (V), formula (VI),formula (VII), formula (VIII), formula (IX), formula (X), formula (XI),formula (XII) and formula (XIII):

(SEQ ID NO: 1) X₁X₂X₃X₄X₅GVX₆AKAGVX₇NX₈FKSESY (I) (SEQ ID NO: 2)(X₉)_(n)GVX₁₀AKAGVX₁₁NX₁₂FKSESY (II) (SEQ ID NO: 3)YKX₁₃LRRX₁₄APRWDX₁₅PLRDPALRX₁₆X₁₇L (III) (SEQ ID NO: 4)YKX₁₈LRR(X₁₉)_(n)PLRDPALRX₂₀X₂₁L (IV) (SEQ ID NO: 5)IKLSGGVQAKAGVINMDKSESM (V) (SEQ ID NO: 6) IKLSGGVQAKAGVINMFKSESY (VI)(SEQ ID NO: 7) IKLSGGVQAKAGVINMFKSESYK (VII) (SEQ ID NO: 8)GVQAKAGVINMFKSESY (VIII) (SEQ ID NO: 9) GVRAKAGVRNMFKSESY (IX)(SEQ ID NO: 10) GVRAKAGVRN(Nle)FKSESY (X) (SEQ ID NO: 11)YKSLRRKAPRWDAPLRDPALRQLL (XI) (SEQ ID NO: 12) YKSLRRKAPRWDAYLRDPALRQLL(XII) (SEQ ID NO: 13) YKSLRRKAPRWDAYLRDPALRPLL (XIII)wherein

-   -   X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄,        X₁₅, X₁₈ and X₁₉ are independently chosen from any amino acid;    -   X₁₆, X₁₇, X₂₀ and X₂₁ are independently chosen from Q, P, Y, I        and L;    -   n is 0, 1, 2, 3, 4 or 5,    -   when X₉ is present more than once, each of said X₉ is        independently chosen from any amino acid;    -   when X₁₉ is present more than once, each of said X₉ is        independently chosen from any amino acid

and wherein at least one protecting group and/or at least one labellingagent is optionally connected to said peptide at an N- and/or C-terminalend.

For example, the peptide compound is a peptide compound that comprises:

(SEQ ID NO: 1) X₁X₂X₃X₄X₅GVX₆AKAGVX₇NX₈FKSESY (I) (SEQ ID NO: 2)(X₉)_(n)GVX₁₀AKAGVX₁₁NX₁₂FKSESY (II) (SEQ ID NO: 3)YKX₁₃LRRX₁₄APRWDX₁₅PLRDPALRX₁₆X₁₇L (III) (SEQ ID NO: 4)YKX₁₈LRR(X₁₉)_(n)PLRDPALRX₂₀X₂₁L (IV) (SEQ ID NO: 5)IKLSGGVQAKAGVINMDKSESM (V) (SEQ ID NO: 6) IKLSGGVQAKAGVINMFKSESY (VI)(SEQ ID NO: 7) IKLSGGVQAKAGVINMFKSESYK (VII) (SEQ ID NO: 8)GVQAKAGVINMFKSESY (VIII) (SEQ ID NO: 9) GVRAKAGVRNMFKSESY (IX)(SEQ ID NO: 10) GVRAKAGVRN(Nle)FKSESY (X) (SEQ ID NO: 11)YKSLRRKAPRWDAPLRDPALRQLL (XI) (SEQ ID NO: 12) YKSLRRKAPRWDAYLRDPALRQLL(XII) or (SEQ ID NO: 13) YKSLRRKAPRWDAYLRDPALRPLL (XIII).

For example, the peptide compound is a peptide compound that consistsessentially of:

(SEQ ID NO: 1) X₁X₂X₃X₄X₅GVX₆AKAGVX₇NX₈FKSESY (I) (SEQ ID NO: 2)(X₉)_(n)GVX₁₀AKAGVX₁₁NX₁₂FKSESY (II) (SEQ ID NO: 3)YKX₁₃LRRX₁₄APRWDX₁₅PLRDPALRX₁₆X₁₇L (III) (SEQ ID NO: 4)YKX₁₈LRR(X₁₉)_(n)PLRDPALRX₂₀X₂₁L (IV) (SEQ ID NO: 5)IKLSGGVQAKAGVINMDKSESM (V) (SEQ ID NO: 6) IKLSGGVQAKAGVINMFKSESY (VI)(SEQ ID NO: 7) IKLSGGVQAKAGVINMFKSESYK (VII) (SEQ ID NO: 8)GVQAKAGVINMFKSESY (VIII) (SEQ ID NO: 9) GVRAKAGVRNMFKSESY (IX)(SEQ ID NO: 10) GVRAKAGVRN(Nle)FKSESY (X) (SEQ ID NO: 11)YKSLRRKAPRWDAPLRDPALRQLL (XI) (SEQ ID NO: 12) YKSLRRKAPRWDAYLRDPALRQLL(XII) or (SEQ ID NO: 13) YKSLRRKAPRWDAYLRDPALRPLL (XIII).

For example, the peptide compound is a peptide compound that consistsof:

(SEQ ID NO: 1) X₁X₂X₃X₄X₅GVX₆AKAGVX₇NX₈FKSESY (I) (SEQ ID NO: 2)(X₉)_(n)GVX₁₀AKAGVX₁₁NX₁₂FKSESY (II) (SEQ ID NO: 3)YKX₁₃LRRX₁₄APRWDX₁₅PLRDPALRX₁₆X₁₇L (III) (SEQ ID NO: 4)YKX₁₈LRR(X₁₉)_(n)PLRDPALRX₂₀X₂₁L (IV) (SEQ ID NO: 5)IKLSGGVQAKAGVINMDKSESM (V) (SEQ ID NO: 6) IKLSGGVQAKAGVINMFKSESY (VI)(SEQ ID NO: 7) IKLSGGVQAKAGVINMFKSESYK (VII) (SEQ ID NO: 8)GVQAKAGVINMFKSESY (VIII) (SEQ ID NO: 9) GVRAKAGVRNMFKSESY (IX)(SEQ ID NO: 10) GVRAKAGVRN(Nle)FKSESY (X) (SEQ ID NO: 11)YKSLRRKAPRWDAPLRDPALRQLL (XI) (SEQ ID NO: 12) YKSLRRKAPRWDAYLRDPALRQLL(XII) or (SEQ ID NO: 13) YKSLRRKAPRWDAYLRDPALRPLL (XIII).

According to another aspect, there is provided a peptide compound thatcomprises a compound chosen from compounds of formula (I), formula (II),formula (III), formula (IV), formula (V), formula (VI), formula (VII),formula (VIII), formula (IX), formula (X), formula (XI), formula (XII)and formula (XIII):

(SEQ ID NO: 1) X₁X₂X₃X₄X₅GVX₆AKAGVX₇NX₈FKSESY (I) (SEQ ID NO: 2)(X₉)_(n)GVX₁₀AKAGVX₁₁NX₁₂FKSESY (II) (SEQ ID NO: 3)YKX₁₃LRRX₁₄APRWDX₁₅PLRDPALRX₁₆X₁₇L (III) (SEQ ID NO: 4)YKX₁₈LRR(X₁₉)_(n)PLRDPALRX₂₀X₂₁L (IV) (SEQ ID NO: 5)IKLSGGVQAKAGVINMDKSESM (V) (SEQ ID NO: 6) IKLSGGVQAKAGVINMFKSESY (VI)(SEQ ID NO: 7) IKLSGGVQAKAGVINMFKSESYK (VII) (SEQ ID NO: 8)GVQAKAGVINMFKSESY (VIII) (SEQ ID NO: 9) GVRAKAGVRNMFKSESY (IX)(SEQ ID NO: 10) GVRAKAGVRN(Nle)FKSESY (X) (SEQ ID NO: 11)YKSLRRKAPRWDAPLRDPALRQLL (XI) (SEQ ID NO: 12) YKSLRRKAPRWDAYLRDPALRQLL(XII) (SEQ ID NO: 13) YKSLRRKAPRWDAYLRDPALRPLL (XIII)

wherein

-   -   X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄,        X₁₅, X₁₈ and X₁₉ are independently chosen from any amino acid;    -   X₁₆, X₁₇, X₂₀ and X₂₁ are independently chosen from Q, P, Y, I        and L;    -   n is 0, 1, 2, 3, 4 or 5,    -   when X₉ is present more than once, each of said X₉ is        independently chosen from any amino acid;    -   when X₁₉ is present more than once, each of said X₉ is        independently chosen from any amino acid

and wherein at least one protecting group and/or at least one labellingagent is optionally connected to said peptide at an N- and/or C-terminalend.

For example, the peptide compound has at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to a peptidecompound chosen from peptide compounds of formula (I), formula (II),formula (III), formula (IV), formula (V), formula (VI), formula (VII),formula (VIII), formula (IX), formula (X), formula (XI), formula (XII)and formula (XIII).

For example, the peptide compound has at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to a peptidecompound represented by formula (I) or SEQ ID NO: 1.

For example, the peptide compound has at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to a peptidecompound represented by formula (II) or SEQ ID NO: 2.

For example, the peptide compound has at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to a peptidecompound represented by formula (III) or SEQ ID NO: 3.

For example, the peptide compound has at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to a peptidecompound represented by formula (IV) or SEQ ID NO: 4.

For example, the peptide compound has at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to a peptidecompound represented by formula (V) or SEQ ID NO: 5.

For example, the peptide compound has at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to a peptidecompound represented by formula (VI) or SEQ ID NO: 6.

For example, the peptide compound has at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to a peptidecompound represented by formula (VII) or SEQ ID NO: 7.

For example, the peptide compound has at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to a peptidecompound represented by formula (VIII) or SEQ ID NO: 8.

For example, the peptide compound has at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to a peptidecompound represented by formula (IX) or SEQ ID NO: 9.

For example, the peptide compound has at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to a peptidecompound represented by formula (X) or SEQ ID NO: 10.

For example, the peptide compound has at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to a peptidecompound represented by formula (XI) or SEQ ID NO: 11.

For example, the peptide compound has at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to a peptidecompound represented by formula (XII) or SEQ ID NO: 12.

For example, the peptide compound has at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to a peptidecompound represented by formula (XIII) or SEQ ID NO: 13.

In one embodiment, n is 0. In one embodiment, n is 1. In one embodiment,n is 2. In one embodiment, n is 3. In one embodiment, n is 4. In oneembodiment, n is 5.

In an embodiment, the peptide compound is represented by formula (I) orformula (II).

In one embodiment, the peptide compound is represented by formula (I) orSEQ ID NO: 1.

In one embodiment, the peptide compound is represented by formula (II)or SEQ ID NO: 2.

In an embodiment, the peptide compound is represented by formula (V),formula (VI), formula (VII), formula (VIII), formula (IX) or formula(X).

In one embodiment, the peptide compound is represented by formula (V).

In one embodiment, the peptide compound is represented by formula (VI).

In one embodiment, the peptide compound is represented by formula (VII).

In one embodiment, the peptide compound is represented by formula(VIII).

In one embodiment, the peptide compound is represented by formula (IX).

In one embodiment, the peptide compound is represented by formula (X).

In one embodiment, the peptide compound is represented by formula (III)or formula (IV).

In one embodiment, the peptide compound is represented by formula (III).

In one embodiment, the peptide compound is represented by formula (IV).

In one embodiment, the peptide compound is represented by formula (XI),formula (XII) or formula (XIII).

In one embodiment, the peptide compound is represented by formula (XI).

In one embodiment, the peptide compound is represented by formula (XII).

In one embodiment, the peptide compound is represented by formula(XIII).

In one embodiment, the peptide is represented by the amino acid sequenceof SEQ ID NO: 1. In one embodiment, the peptide is represented by theamino acid sequence of SEQ ID NO: 2. In one embodiment, the peptide isrepresented by the amino acid sequence of SEQ ID NO: 3. In oneembodiment, the peptide is represented by the amino acid sequence of SEQID NO: 4. In one embodiment, the peptide is represented by the aminoacid sequence of SEQ ID NO: 5. In one embodiment, the peptide isrepresented by the amino acid sequence of SEQ ID NO: 6. In oneembodiment, the peptide is represented by the amino acid sequence of SEQID NO: 7. In one embodiment, the peptide is represented by the aminoacid sequence of SEQ ID NO: 8. In one embodiment, the peptide isrepresented by the amino acid sequence of SEQ ID NO: 9. In oneembodiment, the peptide is represented by the amino acid sequence of SEQID NO: 10. In one embodiment, the peptide is represented by the aminoacid sequence of SEQ ID NO: 11. In one embodiment, the peptide isrepresented by the amino acid sequence of SEQ ID NO: 12. In oneembodiment, the peptide is represented by the amino acid sequence of SEQID NO: 13.

In one embodiment, at least one protecting group is connected to saidpeptide at an N- and/or C-terminal end.

In one embodiment, a succinyl group is connected to the peptidecompound. For example, the peptide compound has the sequence ofSuccinyl-IKLSGGVQAKAGVINMFKSESY, corresponding to SEQ ID NO: 6 andhaving a succinyl group attached thereto at the N-terminal end.

In one embodiment, an acetyl group is connected to the peptide compound.For example, the peptide compound has the sequence ofAcetyl-GVRAKAGVRNMFKSESY (SEQ ID NO: 14). For example, the peptidecompound has the sequence of Acetyl-GVRAKAGVRN(Nle)FKSESY (SEQ ID NO:15). For example, the peptide compound has the sequence ofAcetyl-YKSLRRKAPRWDAPLRDPALRQLL (SEQ ID NO: 16). For example, thepeptide compound has the sequence of Acetyl-YKSLRRKAPRWDAYLRDPALRQLL(SEQ ID NO: 17). For example, the peptide compound has the sequence ofAcetyl-YKSLRRKAPRWDAYLRDPALRPLL (SEQ ID NO: 18).

In one embodiment, at least one labelling agent is connected to saidpeptide at an N- and/or C-terminal end.

The person skilled in the art will understand that commonly usedlabelling agents can be used. For example, the labelling agent is avitamin. For example, the labelling agent is biotin.

In one embodiment, the peptide compound is biotinylated. For example,the peptide compound has the sequence ofIKLSGGVQAKAGVINMFKSESYK(Biotin), corresponding to SEQ ID NO: 7 andhaving a biotin molecule attached thereto at the C-terminal end.

In one embodiment, X₁₆ is independently chosen from Q, P, Y, I and L.

For example, X₁₆ is Q.

For example, X₁₆ is P.

For example, X₁₆ is Y.

For example, X₁₆ is I.

In one embodiment, X₁₇ is independently chosen from Q, P, Y, I and L.

For example, X₁₇ is Q.

For example, X₁₇ is P.

For example, X₁₇ is Y.

For example, X₁₇ is I.

In one embodiment, X₂₀ is independently chosen from Q, P, Y, I and L.

For example, X₂₀ is Q.

For example, X₂₀ is P.

For example, X₂₀ is Y.

For example, X₂₀ is I.

In one embodiment, X₂₁ is independently chosen from Q, P, Y, I and L.

For example, X₂₁ is Q.

For example, X₂₁ is P.

For example, X₂₁ is Y.

For example, X₂₁ is I.

In one embodiment, the peptide compound is chosen from:

(SEQ ID NO: 1) X₁X₂X₃X₄X₅GVX₆AKAGVX₇NX₈FKSESY; (SEQ ID NO: 2)(X₉)_(n)GVX₁₀AKAGVX₁₁NX₁₂FKSESY; (SEQ ID NO: 3)YKX₁₃LRRX₁₄APRWDX₁₅PLRDPALRX₁₆X₁₇L; (SEQ ID NO: 4)YKX₁₈LRR(X₁₉)_(n)PLRDPALRX₂₀X₂₁L; (SEQ ID NO: 5) IKLSGGVQAKAGVINMDKSESM;Succinyl-IKLSGGVQAKAGVINMFKSESY(that comprises SEQ ID NO: 6 wherein a succinylgroup is attached thereto at the N-terminal end);

IKLSGGVQAKAGVINMFKSESYK(Biotin)(that comprises SEQ ID NO: 7 wherein a biotinmolecule is attahed thereto at the C-terminal end);

(SEQ ID NO: 8) GVQAKAGVINMFKSESY; (SEQ ID NO: 14)Acetyl-GVRAKAGVRNMFKSESY; (SEQ ID NO: 15) Acetyl-GVRAKAGVRN(Nle)FKSESY;(SEQ ID NO: 16) Acetyl-YKSLRRKAPRWDAPLRDPALRQLL; (SEQ ID NO: 17)Acetyl-YKSLRRKAPRWDAYLRDPALRQLL; and (SEQ ID NO: 18)Acetyl-YKSLRRKAPRWDAYLRDPALRPLL.

In one embodiment, the peptide compounds can be modified at the C-and/or N-terminal by the addition of one or more amino acid residue inorder to obtain or increase preferential binding sites at the peptideterminal end. For example, the amino acid can be cysteine. For example,the amino acid can be lysine.

The peptide compounds described herein can be connected, linked, mixedor conjugated to small molecules, peptides, anticancer peptides,proteins, oligonucleotides, diagnostic agents, imaging or radionuclideagents, large molecules such as monoclonal antibodies, therapeuticagents such as anticancer agents and phytochemicals or to drug deliverysystems including nanoparticles, liposomes, graphene particles loadedwith a therapeutic agent, imaging agent, gene, siRNA. The resultingconjugate compounds can be used as mono- or combined therapies forexample for treating cancer.

Accordingly, a second aspect disclosed herein is a conjugate compoundhaving the formula of A-(B)_(n),

wherein

-   -   n is 1, 2, 3 or 4;    -   A is a peptide compound as defined in any one of claims 1 to 14,        wherein said peptide is optionally protected by a protecting        group; and    -   B is at least one therapeutic agent, wherein B is connected to        A.

A third aspect disclosed herein is a conjugate compound having theformula of A-(B)_(n),

wherein

-   -   n is 1, 2, 3 or 4;    -   A is a peptide compound as defined herein; and    -   B is at least one therapeutic agent, wherein B is connected to A        at a free amine of a lysine residue of said peptide compound,        optionally via a linker, or at an N-terminal position of said        peptide compound, optionally via a linker.

In an embodiment, B is connected to A via a linker, optionally acleavable linker.

Anticancer agents that can be used include for example alkylatingagents, for example nitrogen mustards (Melphalan, Cyclophosphamide,Ifosfamide), Nitrosoureas, Alkylsulfonates, Ethyleneimines, Triazene,Methyl Hydrazines and Platinum Coordination complexes such as Cisplatin,Carboplatin, Oxaliplatin.

For example, antimetabolites such as folate antagonists (such asmethotrexate), purine antagonists and pyrimidine antagonists (such as5-Fluorouracil and cytabarine) can be used as anticancer agents.

For example, natural products can be used as anticancer agents. Suchnatural products include plant products, for example, vinca alkaloids(such as vincristine, vinblastine), taxanes (such as paclitaxel,docetaxel and cabazitaxel), toxins, epipodopyllotoxins (such asetoposide) and camtothecins (such as irinotecan) and microorganismproducts for example antibiotics (such as doxorubicin and bleomycin, andenzymes such as L-asparaginase.

Other anticancer agents that can be used include for example Maytansine,Auristatin, Dolastin, Chalicheamicin, Emtansine, Amanitin,Pyrrolobenzodiazepines, Tubulysins, Hydroxyurea, Imatinib Mesylate,Rituximab, Epirubicin, Bortezomib, Zoledronic Acid, Geftinib,Leucovorin, Pamidronate and Gemcitabine.

For example, hormones and antagonists such as Corticosteroids(Prednisone, Dexamethasone), Estrogens (Ethinyloestradiol),Antiestrogens (Tamoxifen), Progesteron derivatives (Megestrol Acetate),Androgen (Testosterone propionate), Antiandrogen (Flutamide,Bicalutamide), Aromatase inhibitors (Letrozole, Anastrazole), 5-alphareductase inhibitor (Finasteride), GnRH Analogues (Leuprolide,Buserelin) and Growth Hormone, glucagon and insulin inhibitor(Octreotide) can be used as anticancer agents.

For example, the compounds disclosed herein may be connected toanticancer agents used for targeted cancer therapy including for exampletyrosine kinase inhibitors (TKI) (for example imatinib mesylate,gefitinib, erlotinib, sorafenib, sunitinib, dasatinib, lapatinib,nilotinib and bortezomib), antibodies, monoclonal antibodies (mABs) (forexample rituximab, trastuzumab, alemtuzumab, cetuximab, bevacizumab andipilimumab), mechanistic target of rapamycin (mTOR) inhibitors andantibody-drug conjugates (for example trastuzumab emtansine (T-M D1).

For example, the compounds disclosed herein may be connected to peptidesused for peptide-based cancer therapy such as for example buserelin,gonadorelin, goserelin, histrelin, leuprolide, nafarelin, triptorelin,abarelix, cetrorelix, degarelix and ganirelix.

In an embodiment, the therapeutic agent is an anticancer agent.

For example, the anticancer agent is docetaxel, cabazitaxel, paclitaxel,doxorubicin and daunomycin.

In an embodiment, the anticancer agent is docetaxel.

In an embodiment, the conjugate compound is chosen from compounds offormula (XIV), formula (XV), formula (XVI), formula (XVII) and formula(XVIII):

IK(docetaxel)LSGGVQAK(docetaxel)AGVINMFK(docetaxel)SESY  (XIV)

-   -   that comprises the peptide compound having SEQ ID NO: 6 wherein        each lysine residue has a docetaxel molecule connected thereto;

GVQAK(docetaxel)AGVINMFK(docetaxel)SESY  (XV)

-   -   that comprises the peptide compound having SEQ ID NO: 8 wherein        each lysine residue has a docetaxel molecule connected thereto;

GVRAK(docetaxel)AGVRNMFK(docetaxel)SESY  (XVI)

-   -   that comprises the peptide compound having SEQ ID NO: 9 wherein        each lysine residue has a docetaxel molecule connected thereto;

GVRAK(docetaxel)AGVRN(Nle)FK(docetaxel)SESY  (XVII)

-   -   that comprises the peptide compound having SEQ ID NO: 10 wherein        each lysine residue has a docetaxel molecule connected thereto;        and

YK(docetaxel)SLRRK(docetaxel)APRWDAPLRDPALRQL  (XVIII)

-   -   that comprises the peptide compound having SEQ ID NO: 11 wherein        each lysine residue has a docetaxel molecule connected thereto.

In an embodiment, the conjugate compound is represented by formula(XIV).

In an embodiment, the conjugate compound is represented by formula (XV).

In an embodiment, the conjugate compound is represented by formula(XVI).

In an embodiment, the conjugate compound is represented by formula(XVII).

In an embodiment, the conjugate compound is represented by formula(XVIII).

In another embodiment, the conjugate compound is chosen from:

Succinyl-IK(docetaxel)LSGGVQAK(docetaxel)AGVINMFK(docetaxel)SESY  (XIX)

-   -   that comprises the peptide compound having SEQ ID NO: 6 wherein        each lysine residue has a docetaxel molecule connected thereto;        and wherein a succinyl group is attached at the N-terminal end;

Acetyl-GVRAK(docetaxel)AGVRNMFK(docetaxel)SESY  (XX)

-   -   that comprises the peptide compound having SEQ ID NO: 14 wherein        each lysine residue has a docetaxel molecule connected thereto;

Acetyl-GVRAK(docetaxel)AGVRN(Nle)FK(docetaxel)SESY  (XXI)

-   -   that comprises the peptide compound having SEQ ID NO: 15 wherein        each lysine residue has a docetaxel molecule connected thereto;        and

Acetyl-YK(docetaxel)SLRRK(docetaxel)APRWDAPLRDPALRQLL  (XXII)

-   -   that comprises the peptide compound having SEQ ID NO: 16 wherein        each lysine residue has a docetaxel molecule connected thereto.

In an embodiment, the conjugate compound is represented by formula(XIX).

In an embodiment, the conjugate compound is represented by formula (XX).

In an embodiment, the conjugate compound is represented by formula(XXI).

In an embodiment, the conjugate compound is represented by formula(XXII).

In yet another embodiment, the anticancer agent is doxorubicin.

In an embodiment, the conjugate compound is chosen from compounds offormula (XXIII), formula (XXIV) and formula (XXV):

GVQAK(doxorubicin)AGVINMFK(doxorubicin)SESY  (XXIII)

-   -   that comprises the peptide compound having SEQ ID NO: 8 wherein        each lysine residue has a doxorubicin molecule connected        thereto;

GVRAK(doxorubicin)AGVRN(Nle)FK(doxorubicin)SESY  (XXIV)

-   -   that comprises the peptide compound having SEQ ID NO: 10 wherein        each lysine residue has a doxorubicin molecule connected        thereto; and

YK(doxorubicin)SLRRK(doxorubicin)APRWDAPLRDPALRQLL  (XXV)

-   -   that comprises the peptide compound having SEQ ID NO: 11 wherein        each lysine residue has a doxorubicin molecule connected        thereto.

In an embodiment, the conjugate compound is represented by formula(XXIII).

In an embodiment, the conjugate compound is represented by formula(XXIV).

In an embodiment, the conjugate compound is represented by formula(XXV).

For example, the conjugate compound can be chosen from

Acetyl-GVRAK(doxorubicin)AGVRN(Nle)FK(doxorubicin)SESY  (XXVI)

-   -   that comprises the peptide compound having SEQ ID NO: 15 wherein        each lysine residue has a doxorubicin molecule connected        thereto; and

Acetyl-YK(doxorubicin)SLRRK(doxorubicin)APRWDAPLRDPALRQLL  (XXVII)

-   -   that comprises the peptide compound having SEQ ID NO: 16 wherein        each lysine residue has a doxorubicin molecule connected        thereto.

In an embodiment, the conjugate compound is represented by formula(XXVI).

In an embodiment, the conjugate compound is represented by formula(XXVII).

In an embodiment, the anticancer agent is cabazitaxel.

In an embodiment, the conjugate compound is chosen from compounds offormula (XXVIII), formula (XXIX) and (XXX):

GVRAK(cabazitaxel)AGVRNMFK(cabazitaxel)SESY  (XXVIII)

-   -   that comprises the peptide compound having SEQ ID NO: 9 wherein        each lysine residue has a cabazitaxel molecule connected        thereto;

GVRAK(cabazitaxel)AGVRN(Nle)FK(cabazitaxel)SESY  (XXIX)

-   -   that comprises the peptide compound having SEQ ID NO: 10 wherein        each lysine residue has a cabazitaxel molecule connected        thereto;

andYK(cabazitaxel)SLRRK(cabazitaxel)APRWDAPLRDPALRQL  (XXX)

that comprises the peptide compound having SEQ ID NO: 9 wherein eachlysine residue has a cabazitaxel molecule connected thereto. In anembodiment, the conjugate compound is represented by formula (XXVIII).

In an embodiment, the conjugate compound is represented by formula(XXIX).

In an embodiment, the conjugate compound is represented by formula(XXX).

For example, the conjugate compound can be chosen from compounds offormula (XXXI), formula (XXXII) and (XXXIII):

Acetyl-GVRAK(cabazitaxel)AGVRNMFK(cabazitaxel)SESY  (XXXI)

-   -   that comprises the peptide compound having SEQ ID NO: 14 wherein        each lysine residue has a cabazitaxel molecule connected        thereto;

Acetyl-GVRAK(cabazitaxel)AGVRN(Nle)FK(cabazitaxel)SESY  (XXXII)

-   -   that comprises the peptide compound having SEQ ID NO: 15 wherein        each lysine residue has a cabazitaxel molecule connected        thereto; and

Acetyl-YK(cabazitaxel)SLRRK(cabazitaxel)APRWDAPLRDPALRQLL  (XXXIII)

-   -   that comprises the peptide compound having SEQ ID NO: 16 wherein        each lysine residue has a cabazitaxel molecule connected        thereto.

In an embodiment, the conjugate compound is represented by formula(XXXI).

In an embodiment, the conjugate compound is represented by formula(XXXII).

In an embodiment, the conjugate compound is represented by formula(XXXIII).

Other therapeutic agents that can be used include phytochemicals.

In an embodiment, the phytochemical is curcumin.

In an embodiment, the conjugate compound is represented by formula(XXXIV):

GVRAK(curcumin)AGVRN(Nle)FK(curcumin)SESY  (XXXIV)

-   -   that comprises the peptide compound having SEQ ID NO: 10 wherein        each lysine residue has a curcumin molecule connected thereto.

For example, the conjugate compound can is represented by formula(XXXV):

Acetyl-GVRAK(curcumin)AGVRN(Nle)FK(curcumin)SESY  (XXXV)

-   -   that comprises the peptide compound having SEQ ID NO: 15 wherein        each lysine residue has a curcumin molecule connected thereto.

In an embodiment, B, the at least one therapeutic agent, is connected toA, the peptide compound, at said free amine of said lysine residue ofsaid peptide compound, via a linker.

In an embodiment, B, the at least one therapeutic agent, is connected toA, the peptide compound, at said N-terminal position of said peptidecompound, via a linker.

In an embodiment, the linker is chosen from succinic acid and dimethylglutaric acid linker.

For example, the linker is a cleavable linker.

For example, the linker is a non-cleavable linker.

As shown in Example 2, the conjugate compound can comprise a cleavablelinker connected the at least one therapeutic agent to the peptidecompound. For example, the at least one therapeutic agent can bereleased from the peptide compound by the action of esterases on theester bond.

For example, as shown in Example 2 and in FIG. 4, a therapeutic agentcan be conjugated to the peptide compound on free amines available onthe peptide, at the lysine or amino-terminal, by forming a bond such asa peptide bond.

In an embodiment, the conjugate compound comprises 1 molecule of thetherapeutic agent connected to the peptide compound.

In an embodiment, the conjugate compound comprises 2 molecules of thetherapeutic agent connected to the peptide compound.

In an embodiment, the conjugate compound comprises 3 molecules of thetherapeutic agent connected to the peptide compound.

In an embodiment, the conjugate compound comprises 4 molecules of thetherapeutic agent connected to the peptide compound.

In one embodiment, the conjugation of a therapeutic agent to a peptidecompound, thereby forming a conjugate compound, does not alter thepotency of the therapeutic agent.

For example, as shown in Table 4, IC₅₀ values for the Docetaxel-Katanapeptide conjugate is similar to IC₅₀ values for unconjugated docetaxelin ovary, breast and skin cancer cells.

Conjugate compounds herein disclosed can also be used to transporttherapeutic agents into the cell as they are not a substrate of effluxpumps such as the P-glycoprotein membrane transporter pump which pumpsout other therapeutic agents from multi resistant drug cells.

For example, as shown in FIG. 6, the docetaxel-conjugate uptake inMDCK-MDR cells, kidney epithelial cells transfected with human multidrugresistant gene MDR1, is faster and accumulates at higher concentrationscompared to unconjugated docetaxel.

In a further aspect, there is provided a process for preparing theconjugate compound herein disclosed, the process comprising:

-   -   reacting a linker together with said therapeutic agent so as to        obtain an intermediate;    -   optionally purifying said intermediate;    -   reacting said intermediate together with said peptide compound        so as to obtain said conjugate compound; and    -   optionally purifying said conjugate compound;        wherein the therapeutic agent is connected to the peptide        compound at a free amine of a lysine residue or an N-terminal;        and wherein the peptide compound comprises 1, 2, 3 or 4        therapeutic agent molecules connected thereto.

For example, the peptide compound comprises 1 therapeutic agent moleculeconnected thereto. For example, the peptide compound comprises 2therapeutic agent molecules connected thereto. For example, the peptidecompound comprises 3 therapeutic agent molecules connected thereto. Forexample, the peptide compound comprises 4 therapeutic agent moleculesconnected thereto.

For example, the linker is succinic acid.

For example, the linker is a dimethyl glutaric acid linker.

In an embodiment, the peptide compound is protected at said N-terminalprior to reacting with said intermediate.

Examples of the synthesis of conjugate compounds are shown in Examples11 and 12.

For example, a protecting group such as FMOC can be added as aprotecting group to a free amine on the therapeutic agent prior toincorporation with a linker. After its synthesis, the conjugate compoundcan undergo deprotection from the protecting group. For example, theconjugate compound comprising the protecting agent FMOC can bedeprotected using piperidin. The person skilled in the art would readilyunderstand that other known chemical reagents may be used fordeprotection of conjugate compounds.

For example, the N-terminal of the therapeutic agent and/or the peptidecompound can be capped by its acetylation, thereby providing anon-reversible protecting group at the N-terminal.

In an embodiment, the intermediate is activated prior to reacting withsaid peptide compound.

For example, the intermediate is activated prior to reacting with saidcompound with a coupling agent, optionally chosen fromN,N,N′,N′-Tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate(TBTU), (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) (HBTU), and(1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate) (HATU).

For example, the intermediate comprising a therapeutic agent connectedto a linker can be activated with TBTU, a peptide coupling reagent,prior to conjugation with the peptide compound.

In one embodiment, the conjugate compound is purified following itssynthesis.

Compounds disclosed herein may also be used in the context of fusionproteins. For example, a fusion protein can be engineered by fusing acompound herein disclosed, for example a peptide compound, to one ormore proteins, or parts thereof such as functional domains. Fusionproteins can be engineered for example by recombinant DNA technology andexpressed using a protein expression system such as a bacterial ormammalian protein expression system. In some embodiments, peptidelinkers are added between proteins. In other embodiment, the fusionproteins do not comprise linkers connecting the proteins.

Commonly used protein expression systems include those derived frombacteria, yeast, baculovirus/insect, plants and mammalian cells and morerecently filamentous fungi such as the Myceliophthora thermophile.

In addition, in some embodiments, the compound herein described can beassociated, linked, or connected to one or more other compounds to forma multimer such as a dimer, a trimer or a tetramer, as well as branchedpeptides. Such compounds can be connected together, for example via acovalent bond, an atom or a linker. For example, the multimer comprisesmore than one peptide compound and/or more than one conjugate compound.Methods for making multimeric (e.g. dimeric, trimeric) forms ofcompounds are described in U.S. Pat. No. 9,161,988 which is incorporatedherein by reference in its entirety.

Another aspect of the disclosure includes a method of treating a diseasecomprising administrating a therapeutically effective amount of at leastone compound herein disclosed to a subject in need thereof.

For example, there is provided herein a method of treating a cancerinvolving sortilin expression comprising contacting at least one cancercell expressing sortilin with at least one compound herein disclosed.

For example, there is provided herein a method of treating a diseaseinvolving sortilin expression comprising administering to a subject inneed thereof a therapeutically effective amount of at least one compoundherein disclosed.

For example, there is provided herein a method of treating a cancerinvolving expression of at least one receptor chosen from vacuolarprotein sorting 10 (Vps10) family of receptors comprising contacting atleast one cancer cell expressing the at least one receptor with at leastone compound herein disclosed.

For example, there is provided herein a method of treating a diseaseinvolving expression of at least one receptor chosen from vacuolarprotein sorting 10 (Vps10) family of receptors comprising administeringto a subject in need thereof a therapeutically effective amount of atleast one compound herein disclosed.

For example, the at least one receptor is chosen from sortilin, SorL1,SorCS1, SorCS2, and SorCS3.

For example, the at least one receptor plays pleiotropic functions inprotein trafficking and intracellular and intercellular signaling inneuronal and non-neuronal cells.

For example, in a method of a medical treatment involving a therapeuticagent, the improvement wherein the method comprises increasingtolerability of the therapeutic agent administered to a subject in needthereof by administering the therapeutic agent with at least onecompound herein disclosed.

For example, in a method of a medical treatment involving a therapeuticagent, the improvement wherein the method comprises increasingtolerability of the therapeutic agent administered to a subject in needthereof by administering the therapeutic agent conjugated to at leastone compound herein disclosed.

For example, there is provided herein a method of increasingtolerability of a therapeutic agent, comprising:

-   -   obtaining the conjugate compound herein disclosed, wherein the        conjugate compound comprises the therapeutic agent, and    -   administering a therapeutically effective amount of the        conjugate compound to a subject in need thereof.

For example, there is provided herein a method of increasingtolerability of a therapeutic agent, comprising:

-   -   conjugating the therapeutic agent with the peptide compound        herein disclosed to obtain a conjugate compound, and    -   administering a therapeutically effective amount of the        conjugate compound to a subject in need thereof.

For example, there is provided herein a method of increasinganti-proliferation activity of a therapeutic agent, comprising:

-   -   obtaining the conjugate compound herein disclosed, wherein the        conjugate compound comprises the therapeutic agent, and    -   administering a therapeutically effective amount of the        conjugate compound to a subject in need thereof.

For example, there is provided herein a method of increasinganti-proliferation activity of a therapeutic agent, comprising:

-   -   conjugating the therapeutic agent with the peptide compound        herein disclosed to obtain a conjugate compound, and    -   administering a therapeutically effective amount of the        conjugate compound to a subject in need thereof.

For example, the anti-proliferation activity is increased at least10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least90-fold or at least 100-fold compared to an unconjugated therapeuticagent.

For example, the cancer is ovarian cancer, brain cancer, breast cancer,melanoma, colorectal cancer, glioblastoma, liver cancer, lung cancer,prostate cancer, cervical cancer, head cancer, gastric cancer, kidneycancer, endometrial cancer, testis cancer, urothelial cancer, acutelymphoblastic leukemia, acute myeloid leukemia, Hodgkin lymphoma,neuroblastoma, non-Hodgkin lymphoma, soft tissue cancer, bone sarcoma,thyroid cancer, transitional cell bladder cancer, Wilm's tumour, glioma,pancreatic cancer or spleen cancer.

For example, the cancer is a cancer involving sortilin expression.

For example, there is provided herein a method of increasing cellularinternalization of a therapeutic agent, comprising:

-   -   obtaining the conjugate compound herein disclosed, wherein the        conjugate compound comprises the therapeutic agent, and    -   administering a therapeutically effective amount of the        conjugate compound to a subject in need thereof.

For example, there is provided herein a method of increasing cellularinternalization of a therapeutic agent, comprising:

-   -   conjugating the therapeutic agent with the peptide compound        herein disclosed to obtain a conjugate compound, and    -   administering a therapeutically effective amount of the        conjugate compound to a subject in need thereof.

For example, there is provided herein a method of increasing cellularinternalization of a therapeutic agent, comprising:

-   -   conjugating the therapeutic agent with the peptide compound        herein disclosed to obtain a conjugate compound, and    -   contacting at least one cell with the conjugate compound.

Another aspect includes a use of at least one compound herein disclosedfor treating a disease.

A further aspect includes one or more compound herein disclosed fortreating a disease.

In one embodiment, the disease is a cancer.

Another aspect provided is a method of treating a cancer comprisingadministrating a therapeutically effective amount of at least onecompound herein disclosed to a subject in need thereof.

Another aspect includes a use of at least one compound herein disclosedfor treating a cancer.

Yet another aspect includes one or more compound herein disclosed fortreating a cancer.

In one embodiment, the compound is a conjugate compound hereindisclosed.

Cancers that can be treated using the compounds herein disclosedinclude, but are not limited to, hematological cancers and solidcancers, including for example tumours of the ovary, endometrial, skin,brain, spine, breast, colon, small intestine, liver, lung, prostate,head, neck, stomach, bone, thyroid, bladder, kidney, pancreas andspleen.

In one embodiment, the cancer is ovarian cancer.

In one embodiment, the cancer is breast cancer.

In one embodiment, the cancer is brain cancer.

In one embodiment, the cancer is lung cancer.

In one embodiment, the cancer is skin cancer.

In an embodiment, the cancer is a hematological cancer.

In an embodiment, the hematological cancer is a leukemia such as acutemyeloid leukemia (AML), acute lymphocytic leukemia (ALL), chroniclymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML). Inanother embodiment, the hematological cancer is a myeloma. In anembodiment, the hematological cancer is a lymphoma such as non-Hodgkinlymphoma and Hodgkin lymphoma.

In an embodiment, the cancer is a brain cancer. In an embodiment, thebrain cancer is a glioblastoma.

In an embodiment, the cancer is liver cancer. In an embodiment, theliver cancer is hepatocellular adenocarcinoma.

In an embodiment, the cancer is lung cancer. In an embodiment, the lungcancer non-small cell lung cancer.

In an embodiment, the cancer is kidney cancer. In an embodiment, thekidney cancer is Wilm's tumour.

In an embodiment, the cancer is bladder cancer. In an embodiment, thebladder cancer is transitional cell bladder cancer.

In an embodiment, the cancer is chosen from breast cancer, melanomas,colorectal cancer, glioblastoma and hepatocellular adenocarcinoma.

In one embodiment, the conjugate compound induces apoptosis on cancercells.

For example, Katana-drug conjugates induce apoptosis in cancer cellssuch as for example ovarian cancer cells, melanoma cancer cells andbreast cancer cells.

As shown in FIG. 10, docetaxel-conjugates and cabazitaxel conjugates aremore potent than unconjugated docetaxel and cabazitaxel in inducingapoptosis in cancer cells. Similarly, as shown in FIG. 30, the curcuminconjugate induces greater apoptosis in cancer cells compared tonon-conjugated curcumin.

In one embodiment, Katana-drug conjugates are also more potent thanunconjugated therapeutic agent in inducing tumor suppression.

In one embodiment, the compounds disclosed herein can be used to treatcancer, for example in multidrug resistant cancer.

As shown in FIG. 6, the conjugate compounds were accumulated inMDCK-transfected cells with human multidrug resistant gene MDR1 at afaster rate and at higher concentrations in docetaxel conjugatescompared to unconjugated docetaxel.

In one embodiment, the compounds disclosed herein also decreasemigratory capacity of cancer cells.

For example, as shown in FIG. 16, cell migration was evaluated in cancercells incubated with conjugate compounds. The results show thatKatana-drug conjugates strongly decreased the capacity of the cancercells to migrate.

In one embodiment, the compounds disclosed herein can be used to reducetumour growth.

As demonstrated in Example 4, Katana drug conjugates are more effectivethan unconjugated compounds in reducing tumour growth, as measured byquantitating tumour luminescence in a mouse xenograft tumour model.

The compounds disclosed herein may be used to treat diseases wheresortilin/syndecan receptors are expressed and/or involved.

For example, the compounds herein disclosed may be used to treatinflammatory disease (Mortensen, 2014), lysosomal disorders (Coutinho,2012, Prabakaran, 2012) and cardiovascular disease (Kjolby, 2015).

For example, there is provided a use of a conjugate compound hereindisclosed for increasing cellular internalization of the at least onetherapeutic agent.

For example, there is provided a use of at least one compound hereindisclosed for treating a disease involving sortilin expression.

For example, the use is for treating a disease involving expression ofat least one receptor chosen from vacuolar protein sorting 10 (Vps10)family of receptors.

For example, the at least one receptor is chosen from sortilin, SorL1,SorCS1, SorCS2, and SorCS3.

For example, the at least one receptor plays pleiotropic functions inprotein trafficking and intracellular and intercellular signaling inneuronal and non-neuronal cells.

For example, there is provided a use of at least one compound hereindisclosed for treating a cancer.

For example, there is provided a use of a compound herein disclosed inthe manufacture of a medicament for treating cancer.

For example, the cancer is a cancer involving sortilin expression.

For example, the cancer is ovarian cancer, brain cancer, breast cancer,melanoma, colorectal cancer, glioblastoma, liver cancer, lung cancer,prostate cancer, cervical cancer, head cancer, gastric cancer, kidneycancer, endometrial cancer, testis cancer, urothelial cancer, acutelymphoblastic leukemia, acute myeloid leukemia, Hodgkin lymphoma,neuroblastoma, non-Hodgkin lymphoma, soft tissue cancer, bone sarcoma,thyroid cancer, transitional cell bladder cancer, Wilm's tumour, glioma,pancreatic cancer or spleen cancer.

For example, there is provided a use of a compound herein disclosed forselectively targeting cells expressing sortilin.

It is well known that certain anticancer agents are effective howeverare associated with adverse effects. Doxorubicin for example isassociated with cardiotoxicity in a dose-dependent manner. In humans,the maximum cumulative dose of doxorubicin 550 mg/m². A cumulative doseexceeding this threshold is linked to an increased rate ofcardiotoxicity. As mentioned herein, the compounds described hereinselectively target cells expressing sortilin. As such, they offerselective delivery of anticancer agents to cancer cells expressingsortilin, thus decreasing general cellular toxicity caused by anticanceragents, such as doxorubicin.

For example, there is provided a use of a compound herein disclosed, ina drug delivery system.

For example, there is provided a use of a compound herein disclosed, inthe context of a fusion protein, optionally wherein the fusion proteinis engineered by using an expression system, optionally an expressionsystem derived from bacteria, yeast, baculovirus/insect, plant cells,mammalian cells and filamentous fungi, optionally Myceliophthorathermophila fungi.

For example, there is provided a use of a compound herein disclosed, inthe manufacture of a medicament for treating a disease that involvessortiin expression.

For example, there is provided a use of a compound herein disclosed, forincreasing tolerability of a therapeutic agent.

For example, there is provided a use of a compound herein disclosed, forincreasing tolerability of a therapeutic agent.

For example, there is provided a use of a compound herein disclosed, forincreasing anti-proliferation activity of a therapeutic agent.

Another aspect is a library comprising at least two of compounds hereindisclosed.

Yet another aspect is the use of a library herein described foridentifying compounds that modulate a biological target.

As previously mentioned, the compounds herein disclosed may be used inthe context of drug delivery systems. For example, the compounds may beconnected, linked, mixed, adsorbed to the surface of nanoparticles,liposomes, graphene particles loaded with a therapeutic agent Forexample, the compounds can also be connected to the surface via alinker, an atom or a bond.

An aspect herein disclosed is a liposome, graphene or nanoparticlecomprising at least one compound disclosed herein.

Another aspect is a liposome, graphene or nanoparticle coated with atleast one compound disclosed herein.

Another aspect is a liposome, graphene or nanoparticle loaded with atleast one therapeutic agent, gene or siRNA; and the liposome ornanoparticle is coated with at least one compound herein defined. Forexample, the at least one compound can be connected to the surface ofthe liposome or nanoparticle.

In one embodiment, the at least one compound is a peptide compoundherein disclosed. In one embodiment, the at least one compound is aconjugate compound herein disclosed.

Different embodiments of liposomes or nanoparticles can be envisaged bythe person skilled in the art. For example the liposome or nanoparticlecan comprise at least one peptide compound herein disclosed coated onthe surface of the liposome or nanoparticle and a therapeutic agent, forexample an anticancer agent, within the liposome or nanoparticle. Forexample, the liposome or nanoparticle can comprise at least oneconjugate compound herein disclosed coated on the surface of theliposome or nanoparticle and a therapeutic agent, for example ananticancer agent, within the liposome or nanoparticle.

For example, there is provided herein a multimer comprising two or morecompounds herein disclosed.

For example, the two or more compounds herein disclosed are connected toeach other directly or indirectly.

For example, the two or more compounds are directly connected via acovalent bond.

For example, the two or more compounds are indirectly connected via alinker.

For example, the multimer is a dimer, a trimer or a tetramer.

Further embodiments of the present disclosure will now be described withreference to the following Examples. It should be appreciated that theseExamples are for the purposes of illustrating embodiments of the presentdisclosure, and do not limit the scope of the disclosure.

Example 1 Generation of Peptide Compounds

One of the major goals is to determine whether the Katanareceptor-mediated platform could be efficacious against cancer cells byusing peptide-drug conjugate that are aimed towards receptors expressedon these cells. The first family of Katana peptides is derived frombacterial cell penetrant protein whereas the second family is based onthe sortilin ligands, progranulin and neurotensin (Table 1).

TABLE 1Amino acid sequences of Katana peptides of sortilin-binding peptidesderived from a baterial protein (family 1) and from progranulin andneurotensin, two sortilin ligands (family 2) Amino acid sequenceAmino acid length Katana Biopharma Peptide (KBP) Family 1:KBP-101: IKLSGGVQAKAGVINMDKSESM (SEQ ID NO: 5) 22KBP-102: Succinyl-IKLSGGVQAKAGVINMFKSESY 22(that comprises SEQ ID NO: 6 with a succinylgroup attached at the N-terminal end)KBP-103: IKLSGGVQAKAGVINMFKSESYK(Biotin) 23that comprises SEQ ID NO: 7 with biotinconnected thereto at the C-terminal end)KBP-104: GVQAKAGVINMFKSESY (SEQ ID NO: 8) 17KBP-105: Acetyl-GVRAKAGVRNMFKSESY (SEQ ID NO: 14) 17KBP-106 Acetyl-GVRAKAGVRN(Nle)FKSESY (SEQ ID NO: 15) 17Katana Biopharma Peptide (KBP) Family 2:KBP-201: YKSLRRKAPRVVDAPLRDPALRQLL (SEQ ID NO: 11) 24KBP-202: YKSLRRKAPRVVDAYLRDPALRQLL (SEQ ID NO: 12) 24KBP-203: YKSLRRKAPRVVDAYLRDPALRPLL (SEQ ID NO: 13) 24

Surface plasmon resonance (SPR) was first used to investigate whetherthese peptides could be recognized by sortilin. For this approach,biotin was added on the C-terminal end of the Katana-Biopharma peptideduring peptide synthesis. The biotinylated-peptide (KBP-103) was thenimmobilized on a streptavidin sensor chip using recommended proceduresfrom the manufacturer. Increasing concentrations of a soluble form ofsortilin was then injected over the sensor chip. Interactions betweenimmobilized KBP-103 and the receptor sortilin was then monitored in realtime. A representative interaction curve is shown in FIG. 1, and fromthe sensorgram curves the affinity constant as well as Ka and Kd weredetermined and are indicated in Table 2. Affinity constants (K_(D),K_(a) and K_(d)) were extracted from various injections using the BIAevaluation software. Affinity constant (K_(D)) of the Katana peptide forsortilin is in the low nM range indicating that the peptide has a highaffinity for this receptor.

TABLE 2 Affinity constants K_(D), K_(a) and K_(d) K_(D) k_(a) k_(d)2.55*10⁻⁸ M 6.829*10⁵ M⁻¹ s⁻¹ 0.0174 s⁻¹

The expression of sortilin in various cancer cells by Western blots wasalso investigated. Results show that sortilin can be detected in most ofthe cancer cells tested. In some cases, the expression levels of thisreceptor were very high. High expression was found in many breast cancercells, melanomas, colorectal, glioblastoma and hepatocellularadenocarcinoma (FIG. 3). This is in agreement with the literature sincesortilin has been reported to be expressed in different solid tumoursincluding breast, colorectal, lung, prostate and ovarian cancers(Roseli, 2015; Ghaemimanesh, 2014; Hammati, 2009).

Example 2 Generation of Katana-Peptide Drug Conjugates

Docetaxel and Doxorubicin were first chosen for the proof of principlefor anticancer agents, whereas curcumin was selected amongphytochemicals. Docetaxel is a semi-synthetic analogue of paclitaxel, anextract from the bark of the rare Pacific yew tree Taxus brevifolia.This drug has been approved by the FDA (National Cancer Institute) forthe treatment of locally advanced or metastatic breast cancer, head andneck cancer, gastric cancer, hormone-refractory prostate cancer and nonsmall-cell lung cancer. Docetaxel can be used as a single agent or incombination with other chemotherapeutic drugs depending of specificcancer type and stage. Cabazitaxel (previously XRP-6258, trade nameJevtana™) is a semi-synthetic derivative of a natural taxoid. It is amicrotubule inhibitor that was developed by Sanofi-Aventis. It wasapproved by the U.S. FDA for the treatment of hormone-refractoryprostate cancer on Jun. 17, 2010. Doxorubicin is an anthracyclineantitumour antibiotic (note: in this context, this does not mean it isused to treat bacterial infections) closely related to the naturalproduct Daunomycin and, like all anthracyclines, works by intercalatingDNA, with the most serious adverse effect being life-threatening heartdamage (National Cancer Institute). It is approved to be used alone orwith other drugs to treat: acute lymphoblastic leukemia (ALL), acutemyeloid leukemia (AML), breast cancer, gastric (stomach) cancer, Hodgkinlymphoma, neuroblastoma, non-Hodgkin lymphoma, ovarian cancer, smallcell lung cancer, soft tissue and bone sarcomas, thyroid cancer,transitional cell bladder cancer and Wilm's tumour. Curcumin(diferuloylmethane) is a yellow pigment present in the spice turmeric(Curcuma longa) that has been associated with antioxidant,anti-inflammatory, anticancer, antiviral, and antibacterial activitiesas indicated by over 6,000 citations (Hosseini, 2015).

Anticancer agents (e.g. Docetaxel, Cabazitaxel, Doxorubicin) orphytochemicals (e.g. curcumin) can be conjugated on the peptide usingamine conjugation strategies. Briefly, Docetaxel can be conjugated toKatana peptide(s) on free amines available on the peptide (lysine oramino-terminal) by forming a peptide bond (amide bond) withactivated-Docetaxel. In KBP-101, 4 free amines are available for theconjugation, the N-terminal and 3 lysines. Different conjugates cantherefore be generated by the addition of 1, 2, 3 and 4 Docetaxel to thepeptide. Similar conjugation strategies can be used with Doxorubicin andcurcumin (FIGS. 4 and 5).

For example, the following strategy has been used for the conjugation ofdrug to Katana's peptide. The N-terminal was blocked and all the 3 otherconjugation sites were saturated with Docetaxel, thereby forming apeptide drug conjugate of 3 molecules of Docetaxel per peptide molecule.The whole conjugation was analyzed by HPLC and conjugates were confirmedby Mass spectra (MALDI-TOF). Docetaxel could be released by the cleavageof the ester bond by esterases.

Anticancer agents (ex. Docetaxel, Doxorubicin) and phytochemicals (ex.curcumin) are conjugated using a cleavable linker. The native drug couldthen be released from the vector by the action of esterases on the esterbond. Examples for structures of 2 Katana-anticancer agent conjugates (Aand B) and one phytochemical-Katana drug conjugate (C) are presented inFIG. 5.

Different conjugates between Docetaxel, Doxorubicin, CurcuminCabazitaxel and Katana peptides have been generated. These conjugatesare summarized in Table 3 below.

TABLE 3 Products Amino acid sequences Docetaxel-conjugates KBA102 (3:1)Succinyl-IK(Doce)LSGGVQAK(Doce)AGVINMFK(Doce)SESY KBA104 (2:1)GVQAK(Doce)AGVINMFK(Doce)SESY KBA105 (2:1)Acetyl-GVRAK(Doce)AGVRNMFK(Doce)SESY KBA106 (2:1)Acetyl-GVRAK(Doce)AGVRN(Nle)FK(Doce)SESY KBA201 (2:1)Acetyl-YK(Doce)SLRRK(Doce)APRWDAPLRDPALRQLL Doxorubicin-conjugatesKBB104 (2:1) GVQAK(Doxo)AGVINMFK(Doxo)SESY KBB106 (2:1)Acetyl-GVRAK(Doxo)AGVRN(Nle)FK(Doxo)SESY KBB201 (2:1)Acetyl-YK(Doxo)SLRRK(Doxo)APRWDAPLRDPALRQLL Curcumin-conjugatesKBC106 (2:1) Acetyl-GVRAK(Cur)AGVRN(Nle)FK(Cur)SESYCabazitaxel-conjugates KBD105 (2:1) Acetyl-GVRAK(Cab)AGVRNMFK(Cab)SESYKBD106 (2:1) Acetyl-GVRAK(Cab)AGVRN(Nle)FK(Cab)SESY KBD201 (2:1)Acetyl-YK(Cab)SLRRK(Cab)APRWDAPLRDPALRQLL

Example 3 In Vitro Effects of Conjugate Compounds

The effect of the Docetaxel-Katana peptide conjugate on various cellline proliferations was evaluated and compared to unconjugated Docetaxel(Table 4). IC50 values obtained for the Docetaxel-Katana peptideconjugate were very similar to those of Docetaxel in the cancer cellstested. Overall, these results show that the potency of Docetaxel-Katanapeptide conjugate to block cell proliferation in vitro is similar tounconjugated Docetaxel indicating that the potency of anticancer agentsremains unaltered upon their conjugation.

TABLE 4 Effect of Katana-drug conjugates on cell proliferation using the[³H[-Thymidine incorporation assay. IC50 (nM) obtained fromanti-proliferation curves are presented. IC50 (nM) Tissues CellsDocetaxel Cabazitaxel Doxorubicin Ovary ES-2 Docetaxel: 1.30Cabazitaxel: 0.66 Doxorubicin: 73.5 KBA-105: 0.70 KBD-105: 0.66 KBB-106:66.1 KBA-106: 1.27 KBD-106: 0.24 KBB-201: 83.3 KBA-201: 3.48 KBD-201:0.70 Breast MDA-MB-231 Docetaxel: 0.68 Cabazitaxel: 0.44 Doxorubicin:9.7 KBA-105: 1.03 KBD-105: 0.60 KBB-106: 15.9 KBA-106: 0.38 KBD-106:0.45 KBB-201: 17.3 KBA-201: 0.77 KBD-201: 0.89 Skin SK-MEL-28 Docetaxel:0.69 Cabazitaxel: 0.12 Doxorubicin: 79.3 KBA-105: 0.09 KBD-105: 0.86KBB-106: 68.7 KBA-106: 0.07 KBD-106: 0.04 KBB-201: 81.0 KBA-201: 0.43KBD-201: 0.26 A-375 Docetaxel: 0.92 Cabazitaxel: 0.34 Doxorubicin: 11.8KBA-105: 0.43 KBD-105: 0.45 KBB-106: 13.8 KBA-106: 0.13 KBD-106: 0.42KBB-201: 14.2 KBA-201: 0.81 KBD-201: 0.41

In order to determine whether the anticancer drug-Katana-peptideconjugates could also be P-gp substrates, MDCK-transfected cells withhuman MDR1 were used (MDCK-MDR1). As shown in FIG. 6, the accumulationof the P-gp substrate [³H]-Docetaxel increased by 2-fold in the presenceof cyclosporin A (CsA), a P-gp competitive inhibitor. However, the lackof CsA effect on the accumulation of [¹²⁵I]-Docetaxel-Katana peptideconjugate indicates that upon its conjugation to KBP, the drug moiety isnot recognized anymore by P-gp. The latter results confirm that Katanaconjugates bypass efficiently the efflux action of P-gp.

In addition, the uptake of radiolabeled [¹²⁵I]-Katana conjugate(KBA-105) was compared to that of unconjugated radiolabeled[³H]-Docetaxel in the ovarian SKOV3 cancer cells and in SKMEL-28melanoma cancer cells in FIG. 7. Results demonstrate that the conjugateuptake in SKOV3 (FIG. 7A) as well as in SKMEL-28 cells (FIG. 7B) isfaster and accumulates at higher concentrations than the unconjugatedDocetaxel.

The uptake of the Katana conjugate is increased in cells expressing thesortilin receptor. As shown in FIG. 8, the uptake of the KatanaDoxorubicin conjugate (KBB106) was reduced in cells where sortilinexpression was reduced. For example, FIG. 8A shows decreased uptake ofKBB106 in ovarian cancer cells transfected with sortilin siRNA and FIG.8B shows decreased uptake of KBB106 in ovarian cancer cells due topharmacological inhibition with sortilin ligands, namely the Katanapeptide and progranulin.

The effect of Docetaxel and conjugated Docetaxel on KB-peptide onovarian cancer cell death was also evaluated by flow cytometry analysisusing Annexin V/PI staining (FIG. 9). Results indicate that conjugatedDocetaxel induced a higher and sustained cell death compared to the freedrug (FIG. 9A). In order to induce a similar cell death, addition of theP-gp inhibitor Cyclosporine A (CsA) is required (FIG. 9B). These resultsshow that the KBP-Docetaxel conjugate is more potent, in part throughits ability to bypass the P-gp efflux pump.

The effect of increasing concentration of the Docetaxel-Katana peptideconjugate (KBA-105) or Docetaxel concentration on apoptosis of ovarianSKOV3 cancer cells (FIG. 9) after 5 hours of exposure to the drugs wasalso assessed. Results show that the KBA-105 conjugate induces apoptosisof these cancer cells after a relatively short incubation time.

This apoptosis assay was used to screen the conjugates on various cancercells (FIG. 10). Results indicate that after 5 hours, all Katana-drugconjugates induce apoptosis of the tested cancer cell models. Most ofthem are also more potent than the unconjugated parent drugs, Docetaxelor Cabazitaxel.

In order to determine whether the apoptosis induced by the Katana-drugconjugates was associated to receptor-mediated endocytosis, the assaywas performed in the absence or presence of an excess of free peptideand two sortilin ligand neurotensin (NT) or Progranulin (FIG. 11). Theaddition of the free peptide reversed the apoptosis of SKOV3 (FIG. 11A)and SK-MEL28 (FIG. 11B) induced by the conjugate, indicating thatinduction of these cells by KBA-105 is receptor-mediated. Furthermore,the two sortilin ligands, neurotensin and progranulin also inhibit theapoptosis induced by the Katana-drug conjugate, suggesting that sortilinis involved in this receptor-mediated induction of apoptosis.

The impact of KBP products on the migration of SKOV3 ovarian cancercells. These cancer cells were incubated for 2 hours with either freedocetaxel or a Katana-docetaxel conjugate and cell migration was thenmeasured in real time as a function of time using xCELLigence biosensorsystem. This assay reflects the cellular effects of these molecules onSKOV3 ovarian cancer cell functions. As shown in FIG. 12, theKatana-Docetaxel conjugate has a stronger effect on SKOV3 cells,resulting in a stronger inhibition of their migration when compared tofree Docetaxel. Stronger inhibition of cancer cell migration by theconjugated Docetaxel is an indication that the invasion or metastaticpotential of these cancer cells will be more affected by the conjugatethan by unconjugated Docetaxel.

Interestingly, addition of either an excess of free Katana peptide (FIG.13A) or neurotensin (FIG. 13B) strongly reversed the migratory effect ofthe Katana-Docetaxel conjugate. The reduction in cancer cell migrationby free Docetaxel was unaffected by either free Katana-peptide orneurotensin. In addition, it was found that sortilin gene silencing withspecific sortilin siRNA reversed the effect of the Katana-Docetaxelconjugate on cancer cell migration (FIG. 13C). These results support theconcept that the Katana-Docetaxel conjugate has a distinct mechanism ofaction, different from that of the free drug. The fact that the sortilinligand neurotensin significantly reversed the cellular effect of theconjugate further supports the implication of a receptor member of thesortilin family in the conjugate internalization or mechanism of action.

The effect of Docetaxel and KBP-Docetaxel on cell migration after genesilencing of sortilin using specific siRNA or a scrambled siRNA sequencewas evaluated. Under the experimental conditions used, sortilin geneexpression was reduced by about 80%. Results in FIG. 14 clearly showthat the effect of the free Docetaxel on SKOV3 cell migration wasunaffected by the reduction of sortilin expression. In contrast, theeffect of the conjugated-Docetaxel is strongly reduced when sortilinexpression is low. Reduction of sortilin expression using specificsiRNAs reversed the conjugated-Docetaxel cytotoxic effects, but not thatof free Docetaxel in ovarian cancer cells.

This second cellular assay was also used to screen the differentKatana-drug conjugates (FIG. 15). As shown in FIG. 15A, ovarian (SKOV3and ES-2) cancer cells were incubated for 2 hours with the Doxorubicinor conjugated Doxorubicin (KBB106) (2 μM), washed and then cellmigration was performed. Results show that Katana-drug conjugatesstrongly affected the capacity of these cancer cells to migrate. Forexample, KBA-106 almost completely abolished the cellular capacity ofthese cells to migrate indicating that Katana-drug conjugates exert astrong effect against cancer invasion or dispersion of metastases.However, as shown in FIGS. 15B and 15C, when the cancer cells wereincubated with conjugated Doxorubicin (KBB106) in the presence of asortilin ligand (Neurotensin, Katana peptide or progranulin), themigratory effect of the Doxorubicin conjugate was reversed.

Surface plasmon resonance, apoptosis and migration results providedevidences that Katana-peptide and conjugate interact with or require thesortilin receptor. Interestingly, the sortilin receptor has beenreported to be overexpressed in ovarian cancers as compared to normalovarian tissue (FIG. 16A) (Hemmati 2009, Ghaemimanesh 2014). Sortilinwas also shown to be expressed in various human solid tumours such ascolon, prostate, pancreatic and lung. Furthermore, sortilin expressionhas been associated with the aggressiveness of breast cancer (Roseli,2015). Here, it was also observed that this receptor is expressed invarious human brain tumours from grade I to grade IV (FIG. 16B) and invarious human ovarian cancers from grade I to grade IV (FIG. 16C).Overall, since sortilin is involved in the transport of Katana-peptide,these results indicate that Docetaxel-Katana peptide conjugates couldtarget tumours which express the sortilin receptor.

In addition to these results on sortilin expression in ovarian and braintumours, high levels of sortilin have been reported in various humancancers in the “Human protein atlas”. FIG. 17, taken from the websitehttp://www.proteinatlas.org/clearly demonstrates that high sortilinexpression has been detected in biopsies of human cancers includingmelanoma, breast cancer, endometrial and lung cancers.

Example 4 In Vivo Effects of Conjugate Compounds

Sortilin is overexpressed in cancer tissue but nearly undetectable inhealth tissue. As demonstrated in FIGS. 18A and 18B, sortilin expressionin tissues increases as a function of the malignant tissue phenotype. Inparticular, sortilin expression is higher in ovarian metastases.

a) Docetaxel

To evaluate the impact of the drug conjugation to Katana Biopharmapeptide on drug pharmacokinetics and tissue distribution, mice wereinjected with [¹²⁵I]-Docetaxel-Katana peptide conjugate. Plasma wascollected at different times (FIG. 19A). Radioactivity associated withplasma was quantified and pharmacokinetic parameters were determined. Asindicated in the FIG. 19, the half-life of the conjugated Docetaxel is 6hours. This plasmatic half-life is significantly higher than the 1 hrhalf-life reported in the literature for free Docetaxel (Assessmentreport for Docetaxel Teva from the European Medicines Agency). In FIG.20, plasma concentration of Docetaxel-Katana peptide conjugate wascompared to that of unconjugated Docetaxel after iv bolus injection.Results indicate a much higher area under the curve for theDocetaxel-Katana peptide conjugate compared to that of unconjugatedDocetaxel.

For tissue distribution, radiolabeled Katana-drug conjugate (KBA-105)and Docetaxel were administered by iv bolus injections at an equivalentdose of Docetaxel (FIG. 21). At the indicated times (1, 6 and 24 hours),whole body perfusion was performed for 8 min with saline at a flow rateof 8 ml/min after 2, 6 and 24 hours iv bolus injections. Tissues werethen collected and the radioactivity quantified and levels of theradiolabeled conjugate (KBA-105) were quantified. Results show highaccumulation of the conjugate in the lung, liver, spleen and kidneytissues. Furthermore, levels measured for KBA-105 were higher than thoseof Docetaxel in most tissues. To further characterize the difference inlevels of both radiolabeled compounds, AUC1-24 values were calculatedfor the different tissues and compared in Table 5. AUC1-24 ratio betweenKBA-105 and Docetaxel indicate that the Katana-drug conjugates couldaccumulate in higher levels than unconjugated Docetaxel.

TABLE 5 Area under the curve (AUC1-24) for radiolabeled Docetaxel andKatana-drug conjugate (KBA-105) from FIG. 21 tissue distributionresults. Ratio Estimated Ratio (Estimated Docetaxel KBA105 DocetaxelKBA105/ Docetaxel/ Tissue (ng-hr/ml) (ng-hr/ml) (ng-hr/ml) DocetaxelDocetaxel) Brain 34 1643 723 48.3 21.3 Liver 652 39058 17186 60 26.4Lung 614 309075 135993 500 221 Ovary 1523 2431 1070 1.6 0.7 Heart 6566754 2972 10.3 4.5 Mammary 823 2719 1196 3.3 1.5 Kidney 1072 12911 568112.0 5.3 Spleen 457 25911 11401 24.9 25.0

To further evaluate the in vivo efficacy of the Katana-drug conjugate,SKOV3 cancer cells expressing luciferase were implanted in the mouseflank. Mice with similar tumours were treated with either Docetaxel orthe Katana-drug conjugate KBA-105 at an equivalent dose of Docetaxel (10mg/kg/week). Mice treated with Docetaxel received 3 treatments and micetreated with KBA-105 received 5 treatments. Tumour imaging was performedon different days by injecting the luciferase substrate luciferin and byusing the Xtreme imaging system from Carestream.

Imaging results in FIG. 22 show a much lower luminescence on differentdays for the mouse treated with KBA-105 compared to the one treated withDocetaxel. Luminescence was then quantified and plotted as a function ofdays after implantation (FIG. 23). These early-quantitated luminescenceresults suggest that KBA-105 could be more efficacious than unconjugatedDocetaxel to reduce tumour growth in this ovarian animal tumour model.Mice treated with Docetaxel had a body weight loss close to 20% (FIG.22A) whereas the body weight of mice treated with KBA-105 was stillunaffected by the treatments at 20 mg/kg/week (FIG. 22B). Furthermore,body weight of mice treated after 5 treatments with KBA-105 wasunaffected (FIG. 24) whereas the mice treated at an equivalent dose ofDocetaxel was strongly affected after only 3 treatments.

b) Doxorubicin

The effect of the conjugated and unconjugated Doxorubicin was alsoevaluated on ovarian subcutaneous tumors. Mice were implanted in thewith ES-2 ovarian cancer cells. Tumor growth was measured using acaliper. When tumors reached a tumor volume of about 150 mm³, mice weretreated with Doxorubicin or Katana-Doxorubicin conjugate KBB106 at anequivalent dose of Doxorubicin (6 mg/kg/week). The results show that inmice treated with KBB106, the tumor volume remained about the samefollowing the first treatment however in the mice treated withDoxorubicin, the tumor volume increased following the first treatment(FIG. 25A). Similarly, as demonstrated in FIG. 25B, the tumor volumedecreased by 97% in KBB106 treated mice compared to the vehicle groupcompared to a decrease of 43% in Doxorubicin treated mice compared tothe vehicle group. Progression of SKOV3 xenograft tumors in KBB106treated mice was also significantly decreased compared to the controlgroup (FIG. 26A).

Not only was tumor size suppression more effective with conjugatedDoxorubicin, the tolerability of conjugated Doxorubicin was alsosuperior to that of unconjugated Doxorubicin. In FIG. 25C, it is shownthat treatment with KBB106 was continued up to Day 66 post-treatment.Moreover, treatment with KBB106 had little effect on the body weight ofthe mice (FIG. 26B) thus indicating that treatment with conjugatedDoxorubicin is well tolerated.

Residual tumor burden was assessed in Docetaxel and Docetaxel conjugatetreated mice (FIG. 27). Mice were implanted in the flank with MDA-MB231breast cancer cells expressing luciferase. Tumor growth by luminescencewas visualized using an in vivo imaging system from Carestream. Micewere treated with vehicle, Docetaxel (15 mg/kg/week) or KBA106 (50mg/kg/week). After 74 days post treatment, no luminescence wasdetectable in Docetaxel conjugate treated mice (FIGS. 27B and 27C), thussuggesting no residual tumors in the mice.

c) Curcumin

The phytochemical Curcumin, unconjugated and conjugated, was also testedon cancer cell proliferation (FIG. 28). Breast cancer cells (MDA-MB231)were incubated with increasing concentrations of KBC106 or curcumin.After 72 hrs, thymidine incorporation assay was performed to assess theanti-proliferative properties of both molecules. IC50 values wereextracted from the anti-proliferative curves. As shown in FIG. 28A,KBC106 had a stronger anti-proliferation activity (about 100-fold)against these breast cancer cells compared to unconjugated curcumin.Proliferation assay was also performed using different types of cancercells (ovarian, breast, skin and colorectal cancer cells). IC50 valueswere calculated from the anti-proliferation curves. In all cases, theCurcumin conjugate (KBC106) had stronger anti-proliferative activity(9-100 fold) than unconjugated Curcumin, as shown in Table 6 below.

TABLE 6 Anti-proliferative IC50 values of conjugated and unconjugatedCurcumin Curcumin KBC106 Potency Cancer Cell lines IC50 (nM) IC50 (nM)(x-fold) Ovary ES-2 14 433 1 524 10 Breast MDA-MB231 13 373   135 100(Triple negative) HCC-1569 14 750 1 011 15 (HER2+; Herceptin resistantHCC-1954 11 989 1 286 9 (HER2+; Lapatinib resistant) Skin SK-MEL-28 12454   243 51 A-375 20 889   326 64 Colorectal HT-29 51 287 3 605 14

As demonstrated herein, the Curcumin conjugate (KBC106) cellular uptakeis sortilin dependent. As shown in FIG. 29A, cancer cells incubated withconjugated or unconjugated Curcumin were assessed for uptake for bothmolecules using fluorescent live imaging. It was found that cells hadincreased cellular internalization of conjugated Curcumin as opposed tounconjugated Curcumin. In addition, it was shown that sortilin ligands(Neurotensin, progranulin and free Katana peptide) inhibit cellularupdate of conjugated Curcumin (FIGS. 29B and 29C).

In FIG. 30, it was further evaluated whether the Curcumin conjugate(KBC106) induces cancer cell apoptosis. Cancer cells were incubated withKBC106 and curcumin. First column, uptake for both molecules can bemonitored by fluorescent microscopy. Higher accumulation of fluorescencewas detected for KBC1006 indicating a better cell internalization forthe conjugate. Curcumin accumulation was barely visible in these cancercells. In the second column, Alpha-tubulin was detected by fluorescenceand images indicating that KBC106 strongly affects cancer cellmicrotubules whereas curcumin alone has little effect on them. In thethird column, cell nucleus was labelled with the fluorescent dye DAPI.In contrast to unconjugated Curcumin, the KBC106 clearly induced thenucleus fragmentations showing that the conjugate induces apoptosis ofcancer cells whereas Curcumin alone does not. The last column (Merge)shows that KBC106 fluorescence co-localized with the alpha-tubulindetection.

The Curcumin conjugate (KBC106) was also shown to have an inhibitoryeffect on endometrial cancer growth (FIG. 31). Mice were implanted inthe flank with endometrial (MES) cancer cells. Tumor growth was measuredusing a caliper. When tumors reached a volume of around 150 mm³, micewere treated with KBC106 at 60 mg/kg/twice a week. The results at Day 28post-treatments show that KBC1006 inhibits tumor growth by about 62%compared to the vehicle group, as shown in Table 7.

TABLE 7 Inhibitory effect of KBC106 on endometrial tumor growth at day28 At day 28 Volume Inhibition (mm³) (%) Control 1258 — KBC106 473 62

In conclusion, the results describe new applications for drug-Katanapeptide conjugates including anticancer drugs (small molecules,biologics such as mAbs) and phytochemicals. These anticancer drugconjugates remain active in vitro as evidenced upon efficient inhibitionof cell proliferation, and induction of cell toxicity. Importantly, dataobtained with Katana peptide conjugates indicate that the conjugation ofanticancer drugs to the Katana peptide allows them to escape from P-gpaction. Moreover, these results suggest that conjugation of anticancerdrugs or phytochemicals to Katana peptides increases their efficiency invivo by: 1) targeting receptors expressed or overexpressed in cancercells such as sortilin and therefore will potentially reduce the sideeffects of the conjugated drugs, 2) bypassing the P-gp efflux pumpand/or 3) by modifying the pharmacokinetics or bioavailability of thetherapeutic drug. In fact, for example the conjugation of the anticancerdrug docetaxel to the Katana peptide increases the half-life of the freedrug by about 1 hour to about 6 hours. In addition, by specificallytargeting receptor(s), Katana-drug conjugates are better toleratedcompared to unconjugated drugs at an equivalent dose as observed in thein vivo study with KBA-105 and KBB106. Taken together, data described inthe present disclosure suggest that anticancer drugs and phytochemicalsconjugated to Katana peptide(s) may be used against primary tumours suchas ovarian, breast, lung and skin cancers. In particular, theseconjugates may be used against cancers involving expression oroverexpression of sortilin. In addition, because P-gp efflux pump canlimit the accumulation of potential effective drugs in other diseases,conjugation to the Katana peptide may potentially be used in indicationsoutside of oncology. Furthermore, Katana peptides may be conjugated toother types of molecules including anticancer peptides, larger biologics(ex. monoclonal antibodies), siRNA as well as drug delivery systems suchas nanoparticles and liposomes.

Example 5 Cell Proliferation Assay

Cancer cells were cultured in 96-well white plates (Perkin Elmer). Theywere synchronized for 24 hours in serum-deprived medium. Afterincubation of cells with unconjugated drugs (docetaxel, cabazitaxel,doxorubicin) or with drug-Katana peptide conjugates for 2 or 3 days, allmedia was aspirated and cells were pulse-labeled for 4 hours at 37°C./95% O₂/5% CO₂ with media containing 2.5 μCi/mL [methyl-³H] thymidine(Perkin Elmer). Cells were washed, fixed, and dried before addition ofscintillation fluid (Microscint 0, Perkin Elmer). After 24 hours,cell-associated tritium was quantified by counting on a plate reader(TopCount, Perkin Elmer). Incorporated [³H] thymidine was plotted foreach drug concentration.

Example 6

Cell Migration Assay by xCELLigence Biosensor System

Experiments were carried out using the Real-Time Cell Analyser (RTCA)Dual-Plate (DP) Instrument, the xCELLigence system (Roche Diagnostics,QC). This system was used according to the instructions of the supplier.Then, cells (25 000 cells/well) were seeded in serum-free medium onto aCIM-Plates 16 (Roche diagnostics). These plates are similar toconventional Transwells (8-μm pore size) with gold electrode arrays onthe bottom side of the membrane, which provide a real-time measurementof cell migration. Prior to cell seeding, the underside of the wellsfrom the upper chamber was coated with 25 μL of 0.15% gelatin in PBS andincubated for 1 h at 37° C. The lower chamber was filled with serum-freemedium. The upper chamber of each well was filled with 100 μL ofSKOV3-Luciferase cells (2.5×10⁵ cells/mL) pre-treated for 2 hours withor without conjugated-Docetaxel (2 μM) or free Docetaxel (2 μM). After30 min of adhesion, cell migration was monitored every 5 min for 8 h.The impedance value was measured by the RTCA DP Instrument and wasexpressed as an arbitrary unit called the Cell Index which reflectingthe amount of migration-active cells. Each experiment was performed induplicate wells.

Example 7 Iodination of Conjugate

Peptides were iodinated with standard procedures using iodo-beads fromSigma. Katana-peptides were diluted in 0.1M phosphate buffer, pH 6.5(PB). Two iodo-beads were used for each protein. These beads were washedtwice with 3 ml of PB on a whatman filter and re-suspended in 60 μl ofPB. ¹²⁵I (1 mCi) from Amersham-Pharmacia biotech was added to the beadsuspension for 5 min at room temperature. The iodination for eachpeptide was initiated by the addition of 100 μg (80-100 μl). After anincubation of 10 min at room temperature, the free iodine was removed byHPLC.

Example 8 Drug Accumulation in MDCK-MDR1

Cellular uptake of [³H]-Docetaxel and [¹²⁵I]-Docetaxel-Katana peptideconjugate was measured in P-gp-overexpressing MDCK-MDR1 cells, grown in24-well plates. Cells were washed three times with PBS and preincubatedfor 30 min at 37° C. in culture medium without serum with or without theP-gp inhibitor Cyclosporin A (10 μM). Radiolabelled molecules (50 nM)were then added for 60 min. The cells were rapidly washed three timeswith ice-cold PBS and then lysed in 500 μL of 0.1 M NaOH. The amount ofradiolabelled molecules retained in the cells was counted byβ-scintillation counting (Packard model 1900 TR). An aliquot of celllysate was used in parallel to determine cellular protein concentration.

Example 9 Xenograft Tumour Model

SKOV3 cancer cells (2.5×10⁶ cells) expressing luciferase were implantedin the right flank of mice. Tumour growth was monitored using nearinfrared (NiR) imaging system from Carestream by injecting theluciferase substrate luciferin. Mice were treated with Docetaxel andKatana-drug conjugate (KBA-105) at an equivalent dose of Docetaxel (10mg/kg/week). Docetaxel treatment was stopped after 3 injections due tobody weight loss (around −20%). Treatment with KBA-105 was bettertolerated since body weight of mice treated at an equivalent dose wasunaffected even after 5 treatments. Luminescence associated with tumourgrowth of SKOV3/Luc cancer cells was quantified as a function ofpost-treatment days with the instrument software.

Example 10 Pharmacokinetics and Tissue Distribution

The Docetaxel-Katana peptide conjugate was radiolabeled with [¹²⁵I]—using iodobeads. After radiolabeling free iodine was remove. Mice wereinjected with Docetaxel-[¹²⁵I]-Katana peptide conjugate at 5 mg/kg.Plasma was collected at the indicated times. After 1, 2 and 24 hourssome mice were perfused with saline, sacrificed and tissues werecollected. Radioactivity in the plasma and tissues were measured in aradioactivity counter and results were calculated and expressed in termsof μl/ml for the plasma or ng/g of tissues.

Example 11 Synthesis of Docetaxel-Katana Peptide (KBA-106) ConjugateDoceSuOH

DIEA (0.21 ml, 1.2 mmol) was added dropwise to a suspension of Docetaxel(0.81 g, 1.0 mmol) and succinic anhydride (105 mg, 1.05 mmol) in DMSO (5ml) under stirring. The mixture was stirred at room temperature andmonitored by UPLC-MS. After 2 h, the reaction was complete. The solventwas removed, and the resulting residue was dissolved in DCM and loadedon Biotage silica column for purification. DoceSuOH was obtained as awhite powder after lyophilization, UPLC-MS purity >95%.

KBP106-(SuDoce)₂

DIEA (0.234 mmol) was added dropwise to a solution of DoceSuOH (213 mg,0.234 mmol) and TBTU (75 mg, 0.234 mmol) in DMSO (3-4 ml) in order topreactivate the DoceSuOH. The completion of preactivation was monitoredby UPLC-MS, then a solution of KBP106 (120 mg, 0.062 mmol) in DMSO (0.2ml) was added. The mixture was stirred at room temperature. The reactionwas monitored by UPLC-MS until completion. The reaction mixture waspurified using 30RPC resin column and an AKTA purifier system (10% to80% ACN) to give KBP106-(SuDoce)2 (145 mg) as white powder afterlyophilization, UPLC-MS purity >95%.

Example 12 Synthesis of Doxorubicin-Katana Peptide (KBA-106) ConjugateDoxuribicin Fmoc-DmgOH

In order to incorporate the linker Dmg on Doxorubicin, the free primaryamine in the sugar of Doxo needs first to be protected by an Fmoc group.This DoxoFmoc intermediate was purified on Biotage system. DIEA (0.21ml, 1.2 mmol) was then added dropwise to a suspension of DoxoFmoc (0.81g, 1.0 mmol) and dimethyl glutaric (Dmg) anhydride (105 mg, 1.05 mmol)in DMSO (x ml) under stirring. The mixture was stirred at roomtemperature and the reaction was monitored by UPLC-MS. After completion,the solvent was removed, and the resulting residue was dissolved in DCMand loaded on Biotage silica column for purification. DoxoFmoc-DmgOH wasobtained as a reddish powder after lyophilization, UPLC-MS-MS purity>95%.

KBB106-(Dmg-FmocDoxo)₂ Conjugate

DIEA (0.234 mmol) was added dropwise to a solution of DmgOH-FmocDoxo(27.3 mg, 0.03 mmol) and TBTU (9.6 mg, 0.03 mmol) in DMSO (3-4 ml) inorder to preactivate the DmgOH-FmocDoxo. The completion of preactivationwas monitored by UPLC-MS, then a solution of KBP106 (16 mg, 0.008 mmol)in DMSO (0.2 ml) was added. The mixture was stirred at room temperature.The reaction was monitored by UPLC-MS until completion. The reactionmixture was the purified using 30RPC resin column and an AKTA purifiersystem (10% to 80% ACN; 0.1 Formic acid) to giveKBP106-(DmgOH-FmocDoxo)2.

Fmoc Deprotection from KBP106-Dmg-FmocDoxo

Dmg-FmocDoxo (50 mg) was dissolved in 1.5 ml of DMSO and 10 μl ofpiperidine was added. The mixture turned purple instantaneously and theremoval of the Fmoc group from the Doxo moiety was monitored by UPLC-MS.Deprotection was completed in about 10 minutes. To remove the free Fmocgroup and piperidine, the mixture was then loaded directly on a 30RPCresin column for purification using an AKTA purifier system with agradient of 10-80% ACN; 0.1 Formic Acid). The KBP106-Dmg-Doxo wasobtained as a reddish powder, UPLC-MS purity >95%.

Example 13 Synthesis of Curcumin-Katana Peptide (KBA-106) Conjugate

Dmg (1.5 equivalent) was added to a solution of Curcumin (0.5 g) inpyridine (4 ml). The mixture was stirred under reflux at 65° C. and thereaction was monitored by UPLC-MS. After Dmg incorporation, the solventwas removed, and the resulting residue was dissolved in DCM and loadedon Biotage silica column for purification. Cur-DmgOH was obtained as ayellow powder after lyophilization, UPLC-MS purity >95%.

Addition of the NHS Linker to DmgOH

In this second step, the NHS linker was added to DmgOH-Cur. Briefly, a 5fold excess of NHS (224 mg; 1.95 mmol) and EDC (373 mg; 1.95 mmol) wereadded to DmgOH-Cur (200 mg; 0.39 mmol) in DMSO. After the completion ofNHS incorporation, the mixture was directly loaded on 30RPC resin forpurification on an AKTA purifier system (10-80% ACN; 0.1 (YoFA gradient)

KBP106-(DmgCur)₂

Conjugation was performed in DMSO 80% (pH 9.8) by adding a solution ofKBP106 (50 mg; 0.026 mmol) to NHS-DmgCur (38 mg; 0.062 mmol) dissolve inDMSO. The mixture was stirred at room temperature and the conjugationwas monitored by UPLC-MS. The reaction mixture was the purified using30RPC resin column and an AKTA purifier system (10% to 80% ACN; 0.1Formic acid) to give KBP106-(DmgCur)2 as yellow powder afterlyophilization, UPLC-MS purity >95%.

Example 14 Synthesis of Cabazitaxel-Katana Peptide (KBA-106) ConjugateCabazitaxel-SuOH

DIEA (0.21 ml, 1.2 mmol) was added dropwise to a suspension ofCabazitaxel (0.81 g, 1.0 mmol) and succinic anhydride (105 mg, 1.05mmol) in DMSO (x ml) under stirring. The mixture was stirred at roomtemperature and monitored by UPLC-MS. After 2 h, the reaction wascomplete. The solvent was removed, and the resulting residue wasdissolved in DCM and loaded on Biotage silica column for purification.Cabazitaxel-SuOH was obtained as a white powder after lyophilization,UPLC-MS-MS purity >95%.

KBP106-(SuCabazitaxel)₂

DIEA (0.234 mmol) was added dropwise to a solution of CabazitaxelSuOH(219 mg, 0.234 mmol) and TBTU (75 mg, 0.234 mmol) in DMSO (3-4 ml) inorder to preactivate the CabazitaxelSuOH. The completion ofpreactivation was monitored by UPLC-MS, then a solution of KBP106 (120mg, 0.062 mmol) in DMSO (0.2 ml) was added. The mixture was stirred atroom temperature. The reaction was monitored by UPLC-MS untilcompletion. The reaction mixture was purified using 30RPC resin columnand an AKTA purifier system (10% to 80% ACN) to giveKBP106-(SuCabazitaxel)2 (150 mg) as white powder after lyophilization,UPLC-MS-MS purity >95%.

Example 15 Synthesis of Docetaxel-Katana Peptide (KBA-105) Conjugate

A 2 steps process:

1. Addition of the linker on Docetaxel

-   -   Addition of succinic acid (overnight)    -   Purification on Biotage (1 hour)    -   Evaporation of the solvents (30 min)

2. Conjugation

-   -   Activation of the docetaxel with TBTU (5-10 min)    -   Addition of the peptide    -   Reaction in DMF (no need to follow the pH)    -   Reaction time 30 min    -   Purification on 30 RPC followed by lyophilization at −80° C.        Overall yields around 75%

Purity >95%. Example 16 Synthesis of Doxorubicin-Katana Peptide(KBB-106) Conjugate

A 4 steps process:

1. Addition of Fmoc on the Doxorubicin free amine2. Incorporation of the linker on Doxorubicin-Fmoc intermediate

-   -   Dimethyl glutaric acid (DMG) linker    -   Purification of Doxorubicin(Fmoc)-DMG on Akta followed by        lyophilization

3. Conjugation

-   -   Activation of the Doxorubicin-DMG with TBTU (5-10 min)    -   Addition of the peptide    -   Reaction in DMSO (no need to follow the pH)    -   Reaction time 30 min    -   Purification on AKTA (30 RPC resin) followed by lyophilization        4. Deprotection of the Fmoc group with piperidin (5-10 min)        followed by purification        Overall yields around 42%

Purity >95%.

The embodiments of the paragraphs of the present disclosure arepresented in such a manner in the present disclosure so as todemonstrate that every combination of embodiments, when applicable, canbe made. These embodiments have thus been presented in the descriptionin a manner equivalent to making dependent claims for all theembodiments that depend upon any of the preceding claims (covering thepreviously presented embodiments), thereby demonstrating that they canbe combined together in all possible manners. For example, all thepossible combinations, when applicable, between the embodiments of theparagraphs of the present disclosure are hereby covered.

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1-99. (canceled)
 100. A peptide compound having: at least 92% sequenceidentity to a compound of formula (VI), at least 80% sequence identityto a compound of formula (X), or at least 85% sequence identity to acompound of formula (XI): (SEQ ID NO: 6) IKLSGGVQAKAGVINMFKSESY (VI)(SEQ ID NO: 10) GVRAKAGVRN(Nle)FKSESY (X) (SEQ ID NO: 11)YKSLRRKAPRWDAPLRDPALRQLL (XI)

wherein at least one protecting group and/or at least one agent isoptionally connected to said peptide compound at an N-terminal positionof said peptide compound, at a C-terminal position of said peptidecompound, at a free amine of said peptide compound, at a free —SH ofsaid peptide compound, or at a free carboxyl of said peptide compound.101. The peptide compound of claim 100, wherein the peptide compound isrepresented by formula (VI) and consists of the amino acid sequence ofSEQ ID NO:
 6. 102. The peptide compound of claim 100, wherein thepeptide compound is represented by formula (VII) and consists of theamino acid sequence of SEQ ID NO:
 7. 103. The peptide compound of claim100, wherein the peptide compound is represented by formula (VIII) andconsists of the amino acid sequence of SEQ ID NO:
 8. 104. The peptidecompound of claim 100, wherein the peptide compound is represented byformula (IX) and consists of the amino acid sequence of SEQ ID NO: 9.105. The peptide compound of claim 100, wherein the peptide compound isrepresented by formula (X) and consists of the amino acid sequence ofSEQ ID NO:
 10. 106. The peptide compound of claim 100, wherein thepeptide compound is represented by formula (XI) and consists of theamino acid sequence of SEQ ID NO:
 11. 107. The peptide compound of claim100, wherein the peptide compound is represented by formula (XII) andconsists of the amino acid sequence of SEQ ID NO:
 12. 108. The peptidecompound of claim 100, wherein the peptide compound is represented byformula (XIII) and consists of the amino acid sequence of SEQ ID NO: 13.109. The peptide compound of claim 100, wherein the peptide compound hasat least 90% sequence identity to the compound of formula (X) or formula(XI).
 110. The peptide compound of claim 100, wherein the peptidecompound is connected to the at least one agent.
 111. The peptidecompound of claim 110, wherein the peptide compound is connected to theat least one agent by a linker.
 112. The peptide compound of claim 100,wherein the peptide compound is connected to at least one protectinggroup.
 113. The peptide compound of claim 112, wherein the at least oneprotecting group is acetyl or succinyl.
 114. The peptide compound ofclaim 113, wherein the peptide compound is connected to the at least oneprotecting group to form a peptide compound-protecting group conjugaterepresented by Formula (XXXVIII), Formula (XXXIX), Formula (XXXX),Formula (XXXXI), Formula (XXXXII) or Formula (XXXVI): (SEQ ID NO: 14)Acetyl-GVRAKAGVRNMFKSESY (XXXVIII) (SEQ ID NO: 15)Acetyl-GVRAKAGVRN(Nle)FKSESY (XXXIX) (SEQ ID NO: 16)Acetyl-YKSLRRKAPRWDAPLRDPALRQLL (XXXX) (SEQ ID NO: 17)Acetyl-YKSLRRKAPRWDAYLRDPALRQLL (XXXXI) (SEQ ID NO: 18)Acetyl-YKSLRRKAPRWDAYLRDPALRPLL (XXXXII) Succinyl-IKLSGGVQAKAGVINMFKSESY(XXXVI),

that comprises the peptide compound having SEQ ID NO: 6 connected to asuccinyl group at the N-terminal end.
 115. The peptide compound of claim110, wherein the peptide compound is connected to at least onetherapeutic agent, labelling agent and/or imaging agent.
 116. Thepeptide compound of claim 115, wherein the at least one therapeuticagent is an anticancer agent.
 117. A method of selectively targeting atleast one agent to a sortilin-expressing cell in a population of cells,comprising contacting the peptide compound of claim 110 with thepopulation of cells.
 118. The method of claim 117, wherein the at leastone agent is at least one anticancer agent and the sortilin-expressingcell is a cancer cell.
 119. A method of increasing cellularinternalization of at least one agent in a sortilin-expressing cell,comprising contacting the peptide compound of claim 110 with thesortilin-expressing cell.
 120. The method of claim 119, wherein the atleast one agent is at least one anticancer agent and thesortilin-expressing cell is a cancer cell.